U.S. patent number 6,921,846 [Application Number 09/555,349] was granted by the patent office on 2005-07-26 for antibody production methods relating to disruption of peripheral tolerance in b lympho-cytes.
This patent grant is currently assigned to Duke University. Invention is credited to Thomas F. Tedder.
United States Patent |
6,921,846 |
Tedder |
July 26, 2005 |
Antibody production methods relating to disruption of peripheral
tolerance in B lympho-cytes
Abstract
The subject invention relates a method for the production of
monoclonal antibodies. The method utilizes an immunized animal
having antibody-producing cells with disrupted peripheral
tolerance. The invention also provides a method for the use of such
monoclonal antibodies, and polyclonal antibodies derived from an
immunized animal having antibody-producing cells with disrupted
peripheral tolerance, for in vitro and in vivo clinical diagnostics
and therapeutics.
Inventors: |
Tedder; Thomas F. (Durham,
NC) |
Assignee: |
Duke University (Durham,
NC)
|
Family
ID: |
34752618 |
Appl.
No.: |
09/555,349 |
Filed: |
August 1, 2000 |
PCT
Filed: |
November 25, 1998 |
PCT No.: |
PCT/US98/25253 |
371(c)(1),(2),(4) Date: |
August 01, 2000 |
PCT
Pub. No.: |
WO99/27963 |
PCT
Pub. Date: |
June 10, 1999 |
Current U.S.
Class: |
800/4; 800/13;
800/18; 800/5; 800/6 |
Current CPC
Class: |
C07K
16/40 (20130101); C07K 16/44 (20130101); C07K
2317/92 (20130101) |
Current International
Class: |
C07K
16/44 (20060101); C07K 16/40 (20060101); C12P
021/00 (); A01K 067/00 (); A01K 067/027 () |
Field of
Search: |
;800/4-18
;424/93.21,184.1 ;435/325,326 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Engel et al, Immunity 1995;3:39-50. .
Nielsen et al, EMBO J 1983;2:115-9. .
Mullins et al. Perspectives series: Molecular medcine in
genetically engineered animals pp. 1557-1562 1996 vol. 97, No. 7.
.
Hammer et al. Genetic engineering of mammalian embryos pp. 269-278
Jul. 1986. .
Wall et al. Transgenic dairy cattle: genetic engineering on a large
scale pp. 2213-2224 1997. .
A Strasser et al., Proc. Natl. Acad. Sci, USA, "Enforced BCL2
expression in B-lymphoid cells prolongs antibody responses and
elicits autoimmune disease," Oct. 1991, vol. 88, pp. 8661-8665.
.
GJ Hammerling et al., Proc. Natl. Sci USA, "Self-tolerance to HLA
focuses the response of immunized HLA-transgenic mice on production
of antibody to precise polymorphic HLA alloantigens," Jan. 1990,
vol. 87, pp. 235-239. .
S Yoshino et al., European Journal of Pharmacology, "Effect of a
monoclonal antibody against interleukin-4 on the induction of oral
tolerance in mice," Oct. 1997, 336: 203-209. .
Bohlen et al., "Cytolysis of Leukemic B-Cells by T-Cells Activated
via Two Bispecific Antibodies," Cancer Research, p. 4310-4314,
(Sep. 15, 1993). .
Burnett et al., "Human Monoclonal Antibodies to Defined Antigens,"
Human Hybridomas and Mab, Plenum Press, p. 113-133, (1985). .
Brink et al., "Immunoglobulin M and D Antigen Receptors are Both
Capable of Mediating B Lymphocyte Activation, Deletion, to Energy
After Interaction with Specific Antigen," J. Exp. Med., p.
991-1005, (Oct. 1992). .
Sato et al., "CD19 Regulates B Lymphocyte Signaling Thresholds
Critical for the Development of B-1 Lineage Cells and
Autoimmunity," J. Immunology, vol. 157, p. 4371-4378, (1996). .
Zhou et al., "Tissue-Specific Expression of the Human CD19 Gene in
Transgenic Mice Inhibits Antigen-Independent B-Lymphocyte
Development," Molecular and Cellular Biology, vol.14 (No. 6), p.
3884-3894, (Jun., 1994). .
PCT International Search Report dated Mar. 2, 1999 for
corresponding PCT application No. PCT/US98/25253. .
Tedder et al., "The CD19-CD21 Complex Regulates Signal Review
Transduction Thresholds Governing Humoral Immunity and
Autoimmunity," Immunity, vol. 6, p. 107-118, (Feb., 1997)..
|
Primary Examiner: Li; Q. Janice
Attorney, Agent or Firm: Jenkins, Wilson & Taylor,
P.A.
Government Interests
GRANT STATEMENT
This invention was made in part from government support under Grant
Number AI-26872 from the National Institute of Health (NIH). The
U.S. Government has certain rights in the invention.
Parent Case Text
PRIORITY APPLICATION INFORMATION
This application is a regular United States patent application
under 37 C.F.R. .sctn. 1.111(a) based on and claiming priority to
U.S. Provisional Application Ser. No. 60/065,975 filed Nov. 28,
1997, the entire contents of which are herein incorporated by
reference.
Claims
What is claimed is:
1. A method for producing a monoclonal antibody specific for an
antigen, the method comprising: (a) providing a transgenic mouse
whose genome comprises a DNA sequence encoding a CD19 operably
linked to a promoter and overexpressing CD19 in antibody-producing
cells, wherein said antibody-producing cells have disrupted
peripheral tolerance; (b) immunizing said transgenic mouse with an
antigen to permit said antibody-producing cells to produce
antibodies to the antigen, wherein said antigen is selected from
the group consisting of an autoantigen and a highly conserved
antigen: (c) removing at least a portion of said antibody-producing
cells from the mouse; (d) forming a hybridoma by fusing one of the
antibody-producing cells with an immortalizing cell wherein the
hybridoma is capable of producing a monoclonal antibody to the
antigen; (e) propagating the hybridoma; and (f) harvesting the
monoclonal antibodies produced by the hybridoma.
2. The method of claim 1, wherein said monoclonal antibodies
comprise antibodies having an affinity constant of greater than
1.times.10.sup.5 liters per mole for said antigen.
Description
TECHNICAL FIELD OF THE INVENTION
The subject invention relates generally to a method for the
production of monoclonal antibodies. More particularly, the subject
invention utilizes an animal having antibody-forming cells, such as
B lymphocytes, with disrupted peripheral tolerance. Preferably, the
animal comprises a transgenic animal. The invention also provides a
method for the use of such monodonal antibodies, and polyclonal
antibodies derived from an animal having antibody-forming cells,
such as B lymphocytes, with disrupted peripheral tolerance, for in
vitro and in vivo clinical diagnostics and therapeutics.
The publications and other materials used herein to illuminate the
background of the invention, and in particular cases, to provide
additional details respecting the practice, are incorporated herein
by reference, and for convenience, are referenced by author and
date in the following text, and respectively group in the appended
list of references.
Table of Abbreviations Ab--antibody AFC--antibody-forming cell
Ag--antigen ALP--alkaline phosphatase BSA--bovine serum albumin
Btk--Bruton's tyrosine kinase C--complement, usually followed by a
number from 1 to 9 when referencing the factors of the complement
system in the immune system CD--cluster of differentiation
CD19--cell surface molecule of B lymphocytes CD19KO--CD19-deficient
mice CD19TG--human CD19-transgenic mice CFA--complete Freund's
adjuvant CGG--chicken gamma-globulin CJD--Creutzfeldt-Jakob disease
15B3--a monoclonal antibody against bovine, murine and human prion
protein epitope HAT--hypoxanthine, aminopterin and thymidine
hCD19--human CD19 HPRT--hypoxanthine phosphoribosyl transferase
HRP--horseradish peroxidase Ig--immuoglobulin Ig.sup.HEL
--high-affinity HEL-specific IgM.sup.a and IgD.sup.a -antigen
receptors Lyn--a tyrosine kinase MAb--monoclonal antibody
MHC--major histocompatability complex
NP--(4-hydroxy-3-nitrophenyl)acetyl PNA--peanut agglutinin
PrP--prion protein PrPc--a normal prion protein epitope PrPSc--a
disease related prion protein epitope sHEL--soluble hen egg
lysozyme TSE--transmissible spongiform encephalopathic agents
TUNEL--terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP-biotin nick-end labeling Vav--a protooncogene Xid--X-linked
immunodeficiency--a mutation in Btk
BACKGROUND OF THE INVENTION
Kohler and Milstein are generally credited with having devised the
techniques that successfully resulted in the formation of the first
monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein
(1975) Nature 256:495-497; (1976), Eur. J. Immunol. 6:511-519). By
fusing antibody-forming cells (spleen B-lymphocytes) with myeloma
cells (malignant cells of bone marrow primary tumors), they created
a hybrid cell line arising from a single fused cell hybrid (called
a hybridoma or clone). The hybridoma had inherited certain
characteristics of both the lymphocytes and the myeloma cell lines.
Like the lymphocytes, the hybridoma secreted a single type of
immunoglobulin; moreover, like the myeloma cells, the hybridoma had
the potential for indefinite cell division. The combination of
these two features offered distinct advantages over conventional
antisera.
Antisera derived from vaccinated animals are variable mixtures of
polyclonal antibodies which never can be reproduced identically.
Monoclonal antibodies are highly specific immunoglobulins of a
single type. The single type of immunoglobulins secreted by a
hybridoma is specific to one and only one antigenic determinant, or
epitope, on the antigen, a complex molecule having a multiplicity
of antigenic determinants. For instance, if the antigen is a
protein, an antigenic determinant may be one of the many peptide
sequences (generally 6-7 amino acids in length; Atassi, M. Z.
(1980) Molec. Cell. Biochem. 32:21-43) within the entire protein
molecule. Hence, monoclonal antibodies raised against a single
antigen may be distinct from each other depending on the
determinant that induced their formation. For any given hybridoma,
however, all of the antibodies it produces are identical.
Furthermore, the hybridoma cell line is easily propagated in vitro
or in vivo, and yields monoclonal antibodies in extremely high
concentration.
A monoclonal antibody can be utilized as a probe to detect its
antigen. Thus, monoclonal antibodies have been used in in vitro
diagnostics, for example, radioimmunoassays and enzyme-linked
immunoassays (ELISA), and in in vivo diagnostics, e.g. in vivo
imaging with a radio-labeled monoclonal antibody. Also, a
monoclonal antibody can be utilized as a vehicle for drug delivery
to such antibodies' antigen.
Before a monoclonal antibody can be utilized for such purpose,
however, it is essential that the monoclonal antibody be capable of
binding to the antigen of interest; i.e., the target antigen. This
procedure is carried out by screening the hybridomas that are
formed to determine which hybridomas, if any, produce a monoclonal
antibody that is capable of binding to the target antigen. This
screening procedure can be very tedious in that numerous, for
example, perhaps several thousand monoclonal antibodies may have to
be screened before a hybridoma that produces an antibody that is
capable of binding the target antigen is identified. Accordingly,
there is a need for a method for the production of monoclonal
antibodies that increases the likelihood that the hybridoma will
produce an antibody to the target antigen.
Additionally, the immune systems of conventional animals used in
the production of monoclonal antibodies cannot recognize epitopes
that are highly conserved among vertebrate, and particularly
mammalian species, as "non-self" because of "self" tolerance. The
term "tolerance" is well known in the art and refers to the failure
of an animal's immune system to respond to its own tissues. To the
animal's immune system, a highly conserved epitope appears to be
"self", and no immune response is generated. Therefore,
conventional animals are ineffective in the production of
antibodies against such highly conserved epitopes.
There have been attempts in the prior art to address the problems
found in the production of monoclonal antibodies, particularly with
respect to the streamlining of the screening process for monoclonal
antibodies and, to a certain extent, to the generation of a
monoclonal antibody to an epitope that is highly conservative among
animal species, particularly mammalian species.
One such attempt is described in U.S. Pat. No. 5,223,410 issued to
Gargan et al. on Jun. 29, 1993, assigned to American Biogenetic
Sciences, Inc. This patent describes a method for producing
antibodies using an antigen-free animal. Particularly, it describes
the production of monoclonal antibodies using sterile or germ-free
mice. This patent focuses on the problem of streamlining of the
screening processes for monoclonal antibodies by providing
antigen-free or germ-free animals in which monoclonal antibodies
can be more easily identified.
Korth et al. (1997) "Prion (PrPSc)-Specific Epitope Defined by a
Monoclonal Antibody" (Letter to Nature) Nature 390:74 describes a
monoclonal antibody, 15B3, that can discriminate between the normal
and disease-specific forms of a prion (PrP). Prions are infectious
particles causing transmissible spongiform encephalopathies. 15B3
specifically precipitates bovine, murine, or human PrPSc (the
disease causing form), but not PrPc (the normal form), suggesting
that it recognizes an epitope common to prions from different
species. The 15B3 epitope was mapped as three polypeptide segments
in PrP using immobilized synthetic peptides. However, the
biological activity of this monoclonal antibody, which was produced
from BALB/c mice, is uncharacterized.
In light of the above, a need exists for a method for making
monoclonal antibodies against epitopes that are highly conservative
among vertebrate, and particularly, mammalian species.
SUMMARY OF THE INVENTION
In accordance with the subject invention, a method is provided for
the production of monoclonal antibodies to an antigen
comprising:
(a) immunizing an animal having antibody-forming cells with
disrupted peripheral tolerance with said antigen to permit said
antibody-producing cells to produce antibodies to said antigen;
(b) removing at least a portion of said antibody-producing cells
from said animal:
(c) forming a hybridoma by fusing one of said antibody-producing
cells with an immortalizing cell wherein said hybridoma is capable
of producing a monoclonal antibody to said antigen;
(d) propagating said hybridoma; and
(e) harvesting the monoclonal antibodies produced by said
hybridoma.
The subject invention also provides methods for utilizing a
monoclonal antibody or a polyclonal antibody derived from an animal
having antibody-forming cells with disrupted peripheral
tolerance.
Alternatively, the present invention provides a process of
detecting an antigen, wherein the process comprises immunoreacting
the antigen with an antibody prepared according to the process
described above to form an antibody-polypeptide conjugate, and
detecting the conjugate.
In another aspect, the present invention contemplates a diagnostic
assay kit for detecting the presence of an antigen in a biological
sample, where the kit comprises a first container containing a
first antibody capable of immunoreacting with antigen, with the
first antibody present in an amount sufficient to perform at least
one assay, and wherein the antibody is produced by the process
described above. Preferably, an assay kit of the invention further
comprises a second container containing a second antibody that
immunoreacts with the first antibody, wherein the second antibody
is produced by the processes described above. Thus, more
preferably, the antibodies used in an assay kit of the present
invention are monoclonal antibodies. Even more preferably, the
first antibody is affixed to a solid support. More preferably
still, the first and second antibodies comprise an indicator.
Optionally, the indicator is a radioactive label or an enzyme.
In another embodiment, the present invention contemplates a
diagnostic assay kit for detecting the presence, in a biological
sample, of an antibody immunoreactive with an antigen, the kit
comprising a first container containing the antigen that
immunoreacts with the antibody, with the antigen present in an
amount sufficient to perform at least one assay, and wherein the
antibody is produced by the processes described above.
In another embodiment, the present invention contemplates a method
of producing a non-human animal with an immune system having cells
with a predetermined characteristic. The method comprises the steps
of:
(a) obtaining an animal having immune system cells with a
particular characteristic;
(b) obtaining another animal having immune system cells with either
the same or a different characteristic from the animal of step (a);
and
(c) breeding the animal of step (a) with the animal of step (b) to
produce an animal with an immune system having cells with a
predetermined characteristic.
Accordingly, it is an object of this invention to provide an
improved method for the production of antibodies, particularly
monoclonal antibodies.
It is another object of this invention to provide a method for the
production of antibodies, particularly monoclonal antibodies, using
an animal having antibody producing cells with disrupted peripheral
tolerance.
It is a further object of this invention to provide a method for
the production of antibodies, particularly monoclonal antibodies,
using an animal having antibody producing cells with disrupted
peripheral tolerance.
It is still a further object of this invention to provide a method
of producing a non-human animal with an immune system having cells
with a predetermined characteristic.
Some of the aspects and objects of the invention having been stated
hereinabove, other-aspects and objects will become evident as the
description proceeds, when taken in connection with the
accompanying drawings as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts anti-HEL IgM.sup.a antibody levels in Ig.sup.HEL and
sHEL/Ig.sup.HEL mice that overexpress CD19. Each value indicates
serum levels of HEL-specific IgM.sup.a from individual 2-month-old
(2 mo) or 5- to 10-month old (>5 mo) mice measured by ELISA.
Horizontal bars indicating mean anti-HEL antibody concentrations
for each group are provided for reference. The dashed horizontal
line (arrowhead) delimits the 95% confidence interval for the log
normal distribution of anti-HEL antibody levels observed in
unimmunized 2-month-old sHEL/Ig.sup.HEL mice as described in
Materials and Methods.
FIGS. 2A-D depicts humoral immune responses of (A) sHEL/Ig.sup.HEL
and (B) Ig.sup.HEL mice that overexpress CD19 in response to
immunization with HEL. Two-month-old mice were injected i.p. with
HEL or PBS mixed with CFA on days 0 and 21 (arrows), and were bled
at the indicated times. Levels of serum anti-HEL IgM.sup.a
antibodies for individual mice (dots or squares) were determined by
ELISA. Mean antibody levels are shown as solid (hCD19.sup.+/+) or
dashed (hCD19.sup.-/-) lines. The dashed horizontal lines
(arrowhead) delimit the 95% confidence interval for the log normal
distribution of anti-HEL antibody levels observed in unimmunized
sHEL/Ig.sup.HEL mice.
FIGS. 3A-D depicts signal transduction through surface IgM and CD19
in B cells from (A)sHEL/Ig.sup.HEL /hCD19.sup.+/+ or (B) Ig.sup.HEL
/hCD19.sup.+/+ mice. Relative [Ca.sup.++ ].sub.i levels were
assessed by flow cytometry after gating on the B220.sup.+
population of indo-1 loaded splenocytes. Baseline fluorescence
ratios were collected for 1 min before HEL and/or specific
monoclonal antibodies were added (arrows) at final concentrations
of: HEL, 100 ng/ml; anti-mouse CD19, 40 .mu.g/ml; anti-human CD19,
40 .mu.g/ml. An increase in [Ca.sup.++ ].sub.i over time is shown
as an increase in the ratio of indo-1 fluorescence. Values
represent the ratios of fluorescence intensity of cell populations
after treatment relative to the fluorescence intensity of untreated
cells. These results are representative of those obtained from
three littermate pairs of mice.
FIG. 4 depicts affinity measurements of anti-NP antibodies from
hybridomas of CD19TG and CD19KO mice compared with affinities of
antibodies generated wild-type C57BL/6 mice. Representative anti-NP
antibodies were purified and their affinities for NP (Ka) measured
by fluorescence quenching (bars). For comparison, affinities of
anti-NP antibodies generated by B cells isolated from foci (filled
circles) or germinal centers (filled triangles) are shown as
previously described in the art. The days following NP immunization
that the antibodies were isolated from mice is indicated. The thick
vertical line indicates the lower limit of detection in the
fluorescence quinch assay and the thin vertical line indicates the
average Ka for anti-NP antibodies generated by germinal center B
cells.
FIGS. 5A-B depicts reactivity of anti-NP antibodies with self
antigens.
FIG. 5A depicts hybridoma supernatant fluid was assessed for
reactivity with ssDNA by ELISA. Sera from autoimmune
MRL.sup.lpr/lpr mice was used as a positive control. Values
represent mean OD values (.+-.SD) from triplicate wells. Similar
results were obtained in three independent experiments.
FIG. 5B depicts reactivity of purified TG7-83 antibody with ssDNA
compared with two established anti-ssDNA antibodies of the IgG1
isotype (452s.69 and 165s.3g). Reactivity was significantly higher
(p<0.05, *p<0.01) than the negative control antibodies (B1-8
or TG18-161).
DETAILED DESCRIPTION OF THE INVENTION
In the preferred embodiment of this invention transgenic mouse
models for autoreactive B cells provide a mechanism for determining
the role of CD19 signaling in regulating peripheral tolerance in
autoimmunity. The CD19 cell surface molecule regulates signal
transduction events critical for B lymphocyte development and
humoral immunity. Increasing the density of CD19 expression renders
B lymphocytes hyper-responsive to transmembrane signals, and
transgenic mice that over-express CD19 have increased levels of
autoantibodies. The role of CD19 in tolerance regulation and
auto-antibody generation was therefore examined by crossing mice
that overexpress a human CD19 transgene with transgenic mice
expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and
high-affinity HEL-specific IgM.sup.a and IgD.sup.a (Ig.sup.HEL
antigen receptors).
In the preferred model of peripheral tolerance, B cells in
sHEL/Ig.sup.HEL double-transgenic mice are functionally anergic and
do not produce autoantibodies. However, it was found that
overexpression of CD19 in sHEL/Ig.sup.HEL double-transgenic mice
resulted in a breakdown of peripheral tolerance and the production
of anti-HEL antibodies at levels similar to those observed in
Ig.sup.HEL mice lacking the sHEL autoantigen. Therefore, altered
signaling thresholds due to CD19 overexpression resulted in the
breakdown of peripheral tolerance. Thus, CD19 overexpression shifts
the balance between tolerance and immunity to autoimmunity by
augmenting antigen receptor signaling.
This surprising discovery indicates that animals having antibody
producing cells with disrupted peripheral tolerance are useful in
the production of monoclonal antibodies. Transgenic CD19 mice
wherein CD19 is over-expressed experience a breakdown in peripheral
tolerance. This renders B lymphocytes in such mice hyper-responsive
to transmembrane signals. Such B lymphocytes are thus capable of
distinguishing highly conserved epitopes in mammals when such
epitopes are introduced to the mouse as an antigen. The immune
system of a normal mouse would perceive such an epitope as
identical to something that occurs naturally or is native to the
mouse ("self"). In contrast, the immune system of the CD19
over-expressing transgenic mouse, and particularly the B
lymphocytes of the mouse's immune system, recognize the highly
conservative mammalian epitope as a particle foreign to the mouse's
system ("non-self"), which would instigate an immune response.
This immune response is developed to produce monoclonal antibodies
as described more fully herein. The demonstrated ability to break
down peripheral tolerance as described herein and the breeding
experiments described herein provide a method for manipulating the
immune system of an animal such that an animal having an altered
immune system with desired characteristics can be produced, as more
fully described in Example 3.
While the following terms are believed to have well defined
meanings in the art, the following definitions are set forth to
facilitate explanation of the invention.
The term "immune system" includes all the cells, tissues, systems,
structures and processes, including non-specific and specific
categories, that provide a defense against "non-self" molecules,
including potential pathogens, in an animal.
As is well known in the art, the non-specific immune system
includes phagocytositic cells such as neutrophils, monocytes,
tissue macrophages, Kupffer cells, alveolar macrophages and
microglia. The specific immune system refers to the cells and other
structures that impart specific immunity within a host. Included
among these cells are the lymphocytes, particularly the B cell
lymphocytes and the T cell lymphocytes. These cells also include
natural killer (NK) cells. Additionally, antibody-producing cells,
like B lymphocytes, and the antibodies produced by the
antibody-producing cells are also included within the term "immune
system".
The term "tolerance" is meant to refer to an animal's immune
system's failure to respond to its own tissues or to tissues or
molecules so like its own as to be recognized as its own.
The term "peripheral" or "peripheral lymphoid tissues" refer to the
lymph node-, spleen-, or gut-associated lymphoid tissues wherein
cells, such as B lymphocytes, of the immune system are
developed.
Thus, the term "peripheral" in the context of the term "peripheral
tolerance" indicates a tolerance, or failure to recognize an
antigen, by a cell of the immune system, such as a B lymphocyte, in
the peripheral lymphoid tissues wherein such cells usually react
with antigens.
The term "disrupted peripheral tolerance", as used herein and in
the claims, means any manipulation or alteration of the peripheral
tolerance of the antibody-producing cells of the immune system.
Preferably, the term "disrupted peripheral tolerance" is meant to
refer to the break down of peripheral tolerance, which facilities
monoclonal antibody production in accordance with the methods of
the present invention.
The term "anergy" means a condition in which the immune system of
an animal fails to respond to the injection of an antigen. Thus,
the term "peripheral anergy" means a condition in which the
peripheral immune system of an animal fails to respond to the
injection of an antigen.
The term "autoantibody" means an antibody formed against an epitope
native to the animal.
The term "antibody-producing cell" refers to any antibody-producing
cell within the immune system. Preferably, it is meant to refer to
B lymphocytes.
The term "complement" is meant to refer to the non-specific defense
system that is activated by the bonding of antibodies to antigens
and by this means is directed against specific invaders that have
been identified by antibodies. Eleven complement proteins have been
characterized in the field and are generally referred to by those
having ordinary skill in the art as C1-C9. The complement proteins
act generally along a cascade wherein they contribute to (1)
recognition (C1); (2) activation (C4, C2, and C3, in that order);
and (3) attack (C5-C9). During the attack phase, complement
proteins attach to the cell membrane and destroy the victim cell in
a process known as complement fixation. The complement system is
well known in the art and is more fully described in Fox, Human
Physiology, William C. Brown Pub., DuBuque, Iowa (1987).
The term "humoral immunity" is meant to refer to the form of
acquired immunity in which antibody molecules are secreted in
response to antigenic stimulation.
The term "cell-mediated immunity" is meant to refer to the
immunological defense provided by T cell lymphocytes, which come
into close proximity to their victim cells.
The terms "B cell lymphocytes" or "B lymphocytes" are meant to
refer to a type of lymphocyte that can be transformed by antigens
into plasma cells that secrete antibodies, and are thus responsible
for humoral immunity.
The term "T cell lymphocytes" is meant to refer to a type of
lymphocyte that provides cell-mediated immunity, in contrast to B
lymphocytes that provide humoral immunity to the secretion of
antibodies. There are three sub-populations of T cells: cytotoxic,
helper, and suppressor.
The terms "overexpress", "overexpressing" and "overexpressed" refer
to any level of expression of a gene or protein, whether the gene
be a transgene or a normal gene, that exceeds normal or expected
levels of expression by any amount.
Following long-standing patent law convention, the terms "a" and
"an" mean "one or more" when used in this application, including
the claims.
In the following Detailed Description, the use of transgenic mice
which overexpress CD19 is described as a preferred embodiment of
the instant invention. Such transgenic mice have been developed in
the field according to published techniques (see, for example,
Engel et al. (1995) Immunity 3:39-50 and Zhou (1994) Mol. Cell.
Biol. 14:3884-3894). Thus, these mice are conveniently available as
starting materials. However, it also should be noted that there has
been no disclosure of the production of monoclonal antibodies until
the instant disclosure.
Given advances in transgenic animal techniques, which have been
published in the art, it is believed that any animal can be
utilized in the subject invention, including mouse, pig, rat,
rabbit, guinea pig, goat, sheep, primate, and poultry.
Moreover, while CD19 overexpressing transgenic mice are preferred
because the break down in peripheral tolerance of
antibody-producing cells found in these mice, other animals having
a manipulated or altered characteristics in the cells of their
immune system are contemplated to be within the scope of this
invention. Moderate levels of disrupted peripheral tolerance have
been described in the art with respect to the manipulation of CD45
and with respect to the manipulation of LPR mice. But, there has
been no disclosure of the production of monoclonal antibodies until
the instant disclosure. Finally, other particular candidate
characteristics for manipulation are provided in Example 3.
Production of Monoclonal Antibodies
The animal having antibody-forming cells with disrupted peripheral
tolerance is utilized for the production of monoclonal antibodies.
The system can be utilized to produce a monoclonal antibody to any
antigen that the animal not having antibody-forming cells with
disrupted peripheral tolerance could produce. An exemplary list of
antigens appears in U.S. Pat. No. 3,935,074, the contents of which
are herein incorporated by reference.
However, the animal having antibody-forming cells, such as B
lymphocytes, with disrupted peripheral tolerance provides a much
enhanced immune response to the antigen. Thus, one can increase the
likelihood of locating a B-lymphocyte that produces an antibody
that is capable of binding to a specific epitope of the antigen.
This is a major advantage of the subject invention. In addition,
the system having antibody-forming cells with disrupted peripheral
tolerance system is particularly useful for generating a highly
specific antibody for those antigens with numerous epitopes.
Preferably, the system having antibody-forming cells with disrupted
peripheral tolerance is used to generate monoclonal antibodies to
epitopes that are highly conserved among vertebrate and
particularly, mammalian species. Animals not having
antibody-forming cells with disrupted peripheral tolerance do not
typically respond to such highly conserved epitopes because of
self-tolerance. Stated differently, the immune systems of
conventional animals cannot recognize highly conserved epitopes as
"non-self" and therefore cannot produce antibodies against such an
epitope. The immune systems of the animals of the instant
invention, can recognize highly conserved epitopes as "non-self"
because of the antibody-forming cells with disrupted peripheral
tolerance.
The animals of the instant invention can be immunized by standard
techniques. For example, the animal be immunized at least two times
with at least about three weeks between each immunization, followed
by a prefusion booster.
Somatic Cells
Somatic cells of the animal having the potential for producing
antibody and, in particular B lymphocytes, are suitable for fusion
with a B-cell myeloma line. Those antibody-producing cells that are
in the dividing plasmablast stage fuse preferentially. Somatic
cells can be derived from the lymph nodes, spleens and peripheral
blood of primed animals, and the lymphatic cells of choice depend
to a large extent on their empirical usefulness in the particular
fusion system. However, somatic cells derived from the spleen are
generally preferred. Once primed or hyperimmunized, animals having
antibody-forming cells with disrupted peripheral tolerance can be
used as a source of antibody-producing lymphocytes. Mouse
lymphocytes give a higher percentage of stable fusions with the
mouse myeloma lines described herein below. Indeed, mice are the
preferred animals for use in making monoclonal antibodies because
of the availability of excellent cell lines to use as fusion
partners. However, the use of antibody-producing cells from other
animals is also possible. The choice of a particular animal depends
on the choice of antigen, for it is important that the animal have
a B-lymphocyte in its repertoire of B-lymphocytes that can produce
an antibody to such antigen.
Immortalizing Cells
Specialized myeloma cell lines have been developed from lymphocyte
tumors for use in hybridoma-producing fusion procedures (G. Kohler
and C. Milstein (1976) Eur. J. Immunol. 6:511-519; M. Schulman et
al. (1978) Nature 276:269-270). The cell lines have been developed
for at least three reasons. The first reason is to facilitate the
selection of fused myeloma cells. Usually, this is accomplished by
using myelomas with enzyme deficiencies that render them incapable
of growing in certain selective media that support the growth of
hybridomas. The second reason arises from the inherent ability of
lymphocyte tumor cells to produce their own antibodies. The purpose
of using monoclonal techniques is to obtain immortal fused hybrid
cell lines that produce the desired single specific antibody
genetically directed by the somatic cell component of the
hybridoma. To eliminate the production of tumor cell antibodies by
the hybridomas, myeloma cell lines incapable of producing light or
heavy immunoglobulin chains or those deficient in antibody
secretion mechanisms are used. A third reason for selection of
these cell lines is their suitability and efficiency for
fusion.
Several myeloma cell lines can be used for the production of fused
cell hybrids, including NS-1, X63-Ag8, NIS-Ag4/1, MPC11-45.6TG1.7,
X63-Ag8.653, Sp2/O-Agf14, FO, and S194/5XXO.Bu.1., all derived from
mice, and 210-.RCY3.Agl.+B2.3+L derived from rats. (G. J.
Hammerling, U. Hammerling and J. F. Kearnly, eds. (1981),
Monoclonal antibodies and hybridomas, J. L. Turk, eds. Research
Monographs in Immunology, Vol. 3, Elsevier/North Holland Biomedical
Press, New York).
Fusion
Methods for generating hybrids of antibody-producing spleen or
lymph node cells and immortalizing cells generally comprise mixing
somatic cells with immortalizing cells in a proportion which can
vary from about 20:1 to about 1:1 in the presence of an agent or
agents (chemical, viral or electrical) that promote the fusion of
cell membranes. It is often preferred that the same species of
animal serve as the source of the somatic and immortalizing cells
used in the fusion procedure. Fusion methods have been described by
Kohler and Milstein (1975), Nature 256:495-497; (1976), Eur. J.
Immunol. 6:511-519; by Gefter et al. (1977), Somatic Cell Genet
3:231-236 and by Kozbor et al. (1983), Immunology Today, 4:72. The
fusion-promoting agents used by those investigators were Sendai
virus and polyethylene glycol (PEG), respectively.
One can also utilize the recently developed EBV-transformation
technique (Cole et al. (1985), Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Isolation of Clones and Antibody Detection
Fusion procedures usually produce viable hybrids at very low
frequency, about 1.times.10.sup.-5 to 1.times.10.sup.-8. Because of
the low frequency of obtaining viable hybrids, it is essential to
have a means to select fused cell hybrids from the remaining
unfused cells, particularly the unfused myeloma cells. A means of
detecting the desired antibody-producing hybridomas among the other
resulting fused cell hybrids is also necessary.
Generally, the fused cells are cultured in selective media, for
instance HAT medium, which contains hypoxanthine, aminopterin and
thymidine. HAT medium permits the proliferation of hybrid cells and
prevents growth of unfused myeloma cells which normally would
continue to divide indefinitely. Aminopterin blocks de novo purine
and pyrimidine synthesis by inhibiting the production of
tetrahydrofolate. The addition of thymidine bypasses the block in
pyrimidine synthesis, while hypoxanthine is included in the media
so that inhibited cells can synthesize purine using the nucleotide
salvage pathway. The myeloma cells employed are mutants lacking
hypoxanthine phosphoribosyl transferase (HPRT) and thus cannot
utilize the salvage pathway. In the surviving hybrid, the B
lymphocyte supplies genetic information for production of this
enzyme. Since B lymphocytes themselves have a limited life span in
culture (approximately two weeks), the only cells which can
proliferate in HAT media are hybrids formed from myeloma and spleen
cells.
To facilitate screening of antibody secreted by the hybrids and to
prevent individual hybrids from overgrowing others, the mixture of
fused myeloma and B-lymphocytes is diluted in HAT medium and
cultured in multiple wells of microtiter plates. In two to three
weeks, when hybrid clones become visible microscopically, the
supernatant fluid of the individual wells containing hybrid clones
is assayed for specific antibody production.
The assay must be sensitive, simple and rapid. Assay techniques
include radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, and plaque assays.
Cell Propagation and Antibody Production
Once the desired fused cell hybrids have been selected and cloned
into individual antibody-producing cell lines, each cell line can
be propagated in either of two standard ways. A sample of the
hybridoma can be injected into a histocompatible animal of the type
that was used to provide the somatic and myeloma cells for the
original fusion. The injected animal develops tumors secreting the
specific monoclonal antibody produced by the fused cell hybrid. The
body fluids of the animal, such as serum or ascites fluid, can be
tapped to provide monoclonal antibodies in high concentration.
Alternatively, the individual cell lines can be propagated in vitro
in laboratory culture vessels. The culture medium, containing high
concentrations of a single specific monoclonal antibody, can be
harvested by decantation, filtration or centrifugation.
Use of the Monoclonal Antibody
The monoclonal antibodies made by the method of the subject
invention can be utilized in any technique known or to be developed
in the future that utilizes a monoclonal antibody.
A major use of monoclonal antibodies is in an immunoassay, which is
the measurement of the antigen-antibody interaction. Such assays
are generally heterogeneous or homogeneous. In a homogeneous
immunoassay the immunological reaction usually involves the
specific antibody, a labeled analyte, and the sample of interest.
The signal arising from the label is modified, directly or
indirectly, upon the binding of the antibody to the labeled
analyte. Both the immunological reaction and detection of the
extent thereof are carried out in a homogeneous solution.
Immunochemical labels which may be employed include free radicals,
fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
The major advantage of a homogeneous immunoassay is that the
specific antibody need not be separated from the labeled
analyte.
In a heterogeneous immunoassay, the reagents are usually the
specimen, the specific antibody, and means for producing a
detectable signal. The specimen is generally placed on a support,
such as a plate or a slide, and contacted with the antibody in a
liquid phase. The support is then separated from the liquid phase
and either the support phase or the liquid phase is examined for a
detectable signal employing means for producing such signal. The
signal is related to the presence of the analyte in the specimen.
Means for producing a detectable signal include the use of
radioactive labels, fluorescers, enzymes, and so forth. Exemplary
of heterogeneous immunoassays are the radioimmunossay,
immunofluoroescence methods, enzyme-linked immunoassays, and the
like.
For a more detailed discussion of the above immunoassay techniques,
see Enzyme-Immunoassay, by Edward T. Maggio, CRC Press, Inc., Boca
Raton, Fla., (1980). See also, for example, U.S. Pat. Nos.
3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987; 3,867,517;
3,901,654; 3,935,074; 3,984,533; 3,996,345; and 4,098,876, the
contents of each of which are herein incorporated by reference, and
which listing is not intended to be exhaustive.
Another major use of monoclonal antibodies are in vivo imaging and
therapeutics. The monoclonal antibodies can be labeled with
radioactive compounds, for instance, radioactive iodine, and
administered to a patient intravenously. The antibody can also be
labeled with a magnetic probe. NMR can then be utilized to pinpoint
the antigen. After localization of the antibodies at the antigen,
the antigen can be detected by emission tomographical and
radionuclear scanning techniques, thereby pinpointing the location
of the antigen.
By way of illustration, the purified monoclonal antibody is
suspended in an appropriate carrier, e.g., saline, with or without
human albumin, at an appropriate dosage and is administered
intravenously, e.g., by continuous intravenous infusion over
several hours, as in Miller et al., In Hybridomas in Cancer
Diagnosis and Therapy (1982), incorporated herein by reference.
The monoclonal antibodies of subject invention can be used
therapeutically. Antibodies with the proper biological properties
are useful directly as therapeutic agents. Alternatively, the
antibodies can be bound to a toxin to form an immunotoxin or to a
radioactive material or drug to form a radiopharmaceutical or
pharmaceutical. Methods for producing immunotoxins and
radiopharmaceuticals of antibodies are well-known (see, for
example, Cancer Treatment Reports (1984) 68:317-328).
It also is believed that polyclonal antibodies derived from an
animal having antibody-producing cells with disrupted peripheral
tolerance also can be utilized in immunoassays and provide an
improved result as compared to polyclonal antibodies derived from a
conventional animal. Polyclonal antibodies derived from an animal
having antibody-producing cells with disrupted peripheral tolerance
can be made by utilizing such an animal, as described hereinabove,
and immunization techniques, as described hereinabove, followed by
separating the polyclonal antibodies from the animal by
conventional techniques, e.g. by separating the serum from the
animal.
Means for preparing and characterizing antibodies are well known in
the art (See, e.g., Antibodies-A Laboratory Manual, E. Howell and
D. Lane, Cold Spring Harbor Laboratory, 1988). Monoclonal
antibodies can be readily prepared through use of well-known
techniques such as those exemplified in U.S. Pat. No 4,196,265,
herein incorporated by reference.
Pharmaceutical Compositions
In a preferred embodiment, the present invention provides
pharmaceutical compositions comprising a monoclonal antibody
produced by a process of the present invention and a
physiologically acceptable carrier. Such a composition has a
variety of uses, including, for example but not limited to, use as
a delivery agent for a cytotoxic substance as described herein.
A composition of the present invention is typically administered
parenterally in dosage unit formulations containing standard,
well-known nontoxic physiologically acceptable carriers, adjuvants,
and vehicles as desired. The term "parenteral" as used herein
includes intravenous, intramuscular, intraarterial injection, or
infusion techniques.
Injectable preparations, for example sterile injectable aqueous or
oleaginous suspensions, are formulated according to the known art
using suitable dispersing or wetting agents and suspending agents.
The sterile injectable preparation can also be a sterile injectable
solution or suspension in a nontoxic parenterally acceptable
diluent or solvent, for example, as a solution in
1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
can be employed including synthetic mono- or di-glycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
Preferred carriers include neutral saline solutions buffered with
phosphate, lactate, Tris, and the like.
Assay Kits
In another aspect, the present invention contemplates diagnostic
assay kits for detecting the presence of an antigen in biological
samples, where the kits comprise a first container containing a
first antibody capable of immunoreacting with the antigen with the
first antibody present in an amount sufficient to perform at least
one assay, the antibody obtained from an animal having
antibody-producing cells with disrupted peripheral tolerance.
Preferably, the assay kits of the invention further comprise a
second container containing a second antibody that immunoreacts
with the first antibody, the second antibody obtained from an
animal having antibody-producing cells with disrupted peripheral
tolerance. Thus more preferably, the antibodies used in the assay
kits of the present invention are monoclonal antibodies. Even more
preferably, the first antibody is affixed to a solid support. More
preferably still, the first and second antibodies comprise an
indicator, and, preferably, the indicator is a radioactive label or
an enzyme.
The following Examples have been included to illustrate preferred
modes of the invention. Certain aspects of the following Examples
are described in terms of techniques and procedures found or
contemplated by the present inventor to work well in the practice
of the invention. These Examples are exemplified through the use of
standard laboratory practices of the inventor. In light of the
present disclosure and the general level of skill in the art, those
of skill will appreciate that the following Examples are intended
to be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
EXAMPLE 1
CD19-Regulated Signaling Thresholds Control Peripheral Tolerance
and Autoantibody Production in B Lymphocytes
B lymphocyte tolerance to self antigens is achieved by the negative
selection and elimination of immature B cells that express
high-affinity IgM receptors for autoantigens. Goodnow, C. C. (1996)
Proc. Natl. Acad. Sci. USA 93:2264-2271; Hartley et al. (1993) Cell
72:325-335; Nemazee et al. (1989) Nature 337:562-566; Goodnow, C.
C. (1992) Annu. Rev. Immunol. 10:489-518. Negative selection is
antigen receptor-dependent but also relies on established
triggering thresholds for intracellular signals (Goodnow, C. C.
(1996) Proc. Natl. Acad, Sci. USA 93:2264-2271; Klinman, N. R.
(1996) Immunity 5:189-195). If antigen receptor ligation generates
inadequate intracellular signals because of a low affinity for
autoantigens, or the valency or concentration of autoantigen is
low, autoreactive B cells mature and leave the bone marrow but are
rendered functionally anergic. Goodnow, C. C. (1996) Proc. Natl.
Acad. Sci. USA 93:2264-2271; Goodnow, C. C. (1992) Annu. Rev.
Immunol. 10:489-518; Klinman, N. R. (1996) Immunity 5:189-195;
Adelstein et al. (1991) Science 251:1223-1225; Nemazee et al.
(1991) Immunol. Rev. 122:117-132. Intracellular signaling
thresholds are likely to also play a major role in the regulation
and maintenance of peripheral tolerance.
The CD19 cell surface molecule regulates intracellular signaling
thresholds critical for B cell development and humoral immunity.
Tedder et al. (1997) Immunity 6:107-118; Fearon et al. (1996)
Science 272:50-54; Carter et al. (1992) Science 256:105-107;
Dempsey et al. (1996) Science 271:348-350; Engel et al. (1995)
Immunity 3:39-50; Rickert et al. (1995) Nature 376:352-355. B
lymphocytes from mice that overexpress CD19 are hyper-responsive to
antigen receptor crosslinking, which results in serum
immunoglobulin (Ig) levels that are increased by about 40% and
humoral responses that are augmented several fold. Engel et al.
(1995) Immunity 3:39-50; Sato et al. (1995) Proc. Natl. Acad. Sci.
USA 92:11558-11562; Zhou et al. (1994) Mol. Cell. Biol.
14:3884-3894. Based on this, it was expected that CD19
overexpression by autoreactive B cells would either lead to their
augmented negative selection in the bone marrow or result in a more
profound state of peripheral anergy.
Unexpectedly however, C57BL/6 mice that overexpress CD19 have
twofold to fourfold higher levels of anti-DNA autoantibodies and
rheumatoid factor. Tedder et al. (1997) Immunity 6:107-118; Sato et
al. (1996) J. Immunol. 156:4371-4378. Increased autoantibody
production in mice overexpressing CD19 correlates with dramatic
increases in the number of B1 lineage cells. However, since IgG
anti-DNA autoantibodies are preferentially increased in mice that
overexpress CD19, the CD19-induced autoantibodies may alternatively
result from alterations in conventional B-cell tolerance.
Transgenic mouse models for autoreactive B cells (Goodnow, C. C.
(1992) Annu. Rev. Immunol. 10:489-518; Nemazee et al. (1991)
Immunol. Rev. 122:117-132) provide a mechanism for determining the
role of CD19 signaling in regulating peripheral tolerance and
autoimmunity. B cells from transgenic mice expressing a model
autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity
HEL-specific IgM.sup.a and IgD.sup.a (Ig.sup.HEL) antigen receptors
enter the peripheral pool but are anergic to antigen receptor
ligation and produce little, if any, spontaneous HEL-specific
antibody. Goodnow et al. (1988) Nature 334:676-682. Mice that
express a human CD19 (hCD19) transgene provide a model for
examining augmented CD19 function in vivo. Tedder et al. (1997)
Immunity 6:107-118; Sato et al. (1995) Proc. Natl. Acad. Sci. USA
92:11558-11562; Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894;
Sato et al. (1996) J. Immunol. 156:4371-4378; Sato et al. (1997) J.
Immunol. 158:4662-4669) Since hCD19 can replace the function of
mouse CD19 in vivo, hemizygous hCD19.sup.+/- transgenic mice
express cell surface CD19 at a twofold higher density while
hCD19.sup.+/+ transgenic mice express threefold higher densities of
CD19. Sato et al. (1996) J. Immunol. 156:4371-4378; Sato et al.
(1997) J. Immunol 158:4662-4669.
Therefore, sHEL/Ig.sup.HEL double-transgenic mice were crossed with
hCD19 transgenic mice to determine whether tolerance would be
maintained in sHEL/Ig.sup.HEL /hCD19 transgenic mice or
autoantibodies would be generated. CD19 overexpression in
sHEL/Ig.sup.HEL double-transgenic mice resulted in the production
of anti-HEL antibodies at levels similar to those observed in
Ig.sup.HEL mice lacking this model self antigen. Therefore, lowered
signaling thresholds due to CD19 overexpression resulted in the
breakdown of peripheral tolerance in sHEL/Ig.sup.HEL
double-transgenic mice.
Mice. hCD19 transgenic mice (h19-1 line, C57BL/6) were produced as
described in Engel et al. (1995) Immunity 3:39-50 and in Zhou et
al. (1994) Mol. Cell. Biol. 14:3884-3894). In the h19-1 line of
mice, 9-14 copies of the hCD19 transgene are integrated into a
single (or closely linked) site(s). These h19-1 mice used in this
study were backcrossed onto a wild-type C57BL/6 background for 8 to
10 generations without a diminution of hCD19 expression and all
mice express similar levels of cell-surface hCD19. Mice expressing
sHEL (ML5 line)and Ig.sup.HEL (MD4 line) were as described (Goodnow
et al. (1988) Nature 334:676-682; Hartley et al. (1991) Nature
353:765-769). sHEL/Ig.sup.HEL /hCD19 triple-transgenic mice were
generated by appropriate backcrosses of sHEL/Ig.sup.HEL
double-transgenic mice with hCD19.sup.+/+ mice. Transgene
expression was assessed as described in (Engel et al. (1995)
Immunity 3:39-50; Zhou et al. (1994) Mol. Cell. Biol. 14:3884-3894;
Goodnow et al. (1988) Nature 334:676-682; Hartley et al. (1991)
Nature 353:765-769.) Mice were housed in a specific pathogen-free
barrier facility. All studies and procedures were approved by the
Duke University Animal Care and Use Committee.
Immunization of Mice. Two-month-old mice were immunized i.p. with
100 .mu.g of HEL in complete Freund's adjuvant (CFA, Sigma Chemical
Co.) or PBS in CFA at day 0 and were boosted at day 21. Animals
were bled just before the first immunization and 7, 14, and 28 days
later.
Mouse Ig Isotype-specific ELISAs. Serum levels of HEL-specific IgM
allotype a (IgM.sup.a) antibody were measured by ELISA on
HEL-coated plates as described (Goodnow et al. (1989) Nature
342:385-391). Absolute antibody concentrations were determined
relative to a standard curve of HEL-specific IgM.sup.a monoclonal
antibody (E1 clone) generated from an IgHEL transgenic mouse
immunized with HEL. The ELISA sensitivity limit was about 20 ng/ml
of anti-HEL IgM.sup.a antibody.
Immunofluorescence Analysis. Antibodies used in this study
included: FITC-conjugated and biotin-coupled goat anti-mouse IgM
isotype-specific antibodies (Southern Biotechnology Associates,
Inc., Birmingham, Ala.); anti-B220 (CD45RA, RA3-6B2, provided by R.
L. Coffman, DNAX Research Inst., Palo Alto, Calif.), anti-I-A
(M5/114.15.2, American Type Culture Collection (ATCC), Bethesda,
Md., clone TIB120), anti-HSA (M1/69, PharMingen, San Diego,
Calif.), anti-CD5 (53-7.313, ATCC, clone TIB104), anti-B7-2 (GL-1,
PharMingen) and anti-mouse IgM.sup.a (DS-1, PharMingen) monoclonal
antibodies. Phycoerythrin-conjugated streptavidin (Fisher
Scientific, Fair Lawn, N.J.) was used to reveal biotin-coupled
monoclonal antibody staining. Phycoerythrin-conjugated goat
anti-rat IgG antibodies (Caltag, Burlingame, Calif.) were used to
visualize anti-CD5 monoclonal antibody staining. Cells reacting
with biotin-coupled HEL were stained with phycoerythrin-conjugated
streptavidin. Isolated lymphocytes were analyzed on a FACScan.RTM.
flow cytometer (Becton-Dickinson, San Jose, Calif.) as described
(Sato et al. (1996) J. Immunol. 156:4371-4378.)
Measurement of Intracellular Calcium. Splenocytes were isolated,
loaded with indo-1 and stained with FITC-labeled anti-B220
antibodies as described (Sato et al. (1996) Immunity 5:551-562.).
Relative intracellular Ca.sup.++ levels ([Ca.sup.++ ].sub.i) were
assessed by flow cytometry after gating on the B220.sup.+
population of cells. Baseline fluorescence ratios were collected
for 1 min before HEL and/or specific monoclonal antibodies were
added at final concentrations of: HEL, 100 ng/ml; anti-mouse CD19,
40 .mu.g/ml (MB19-1, IgA) (Sato et al. (1996) J. Immunol.
156:4371-4378); and anti-human CD19, 40 .mu.g/ml (HB12b, IgG1)
(Bradbury et al. (1992) J. Immunol. 149:2841-2850.) An increase in
the ratio of indo-1 fluorescence indicates an increase in
[Ca.sup.++ ].sub.i.
Statistical Analysis. All data are shown as mean values.+-.SEM.
Analysis of variance (ANOVA) was used to analyze the data, and the
Student's t test was used to compare population sample means. The
Mann-Whitney test was also used to compare population frequency
distributions. The 95% confidence interval for anti-HEL antibody
levels observed in sHEL/Ig.sup.HEL mice was determined using the
log normal distribution (mean.+-.2 SD) of antibody values with
undetectable levels (<20 ng/ml) assigned the value of 10
ng/ml.
Autoantibodies in sHEL/Ig.sup.HEL /hCD19 transgenic mice. Serum
anti-HEL IgM.sup.a autoantibody levels in Ig.sup.HEL transgenic,
sHEL/Ig.sup.HEL double-transgenic, and sHEL/Ig.sup.HEL /hCD19
triple-transgenic mice were determined to assess the status of B
cell tolerance in each set of mice. Serum antibody levels for each
individual mouse are shown in FIG. 1 and mean autoantibody levels
for each set of mice are provided to simplify discussion of the
results. Two-month-old sHEL/Ig.sup.HEL double transgenic mice
produced very low or undetectable levels of anti-HEL IgM.sup.a
antibodies (mean levels 31 ng/ml) when compared with Ig.sup.HEL
transgenic mice (mean 16,700 ng/ml, FIG. 1). However, 45% (14 of
33) of sHEL/Ig.sup.HEL /hCD19.sup.+/- mice had anti-HEL IgM.sup.a
autoantibody levels (mean 2,430 ng/ml) that were significantly
greater than those found in sHEL/Ig.sup.HEL mice (P.ltoreq.0.001,
FIG. 1). Anti-HEL IgM.sup.a antibody levels were also elevated in
38% (14 of 36) of sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice (mean 10,500
ng/ml, P.ltoreq.0.01, FIG. 1). Autoantibody levels in some
sHEL/Ig.sup.HEL /hCD19 mice were equivalent to those of Ig.sup.HEL
-transgenic mice not expressing sHEL. In fact, overexpression of
CD19 resulted in anti-HEL autoantibody levels in some mice that
were one thousand (1,000)-fold higher than in sHEL/Ig.sup.HEL mice.
By comparison, overexpression of CD19 in Ig.sup.HEL /CD19.sup.+/+
mice resulted in only a fourfold increase in anti-HEL antibody
levels (mean 77,300 ng/ml, FIG. 1). Thus, lowered signaling
thresholds resulting from the overexpression of CD19 abrogated
peripheral anergy in a significant proportion of two-month-old
sHEL/Ig.sup.HEL mice.
The breakdown in peripheral tolerance and the development of
autoantibodies in sHEL/Ig.sup.HEL mice that overexpressed CD19
correlated with mouse age. By five to ten months of age, all
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice produced significantly higher
levels of autoantibodies (mean 144,000 ng/ml) than sHEL/Ig.sup.HEL
mice (300 ng/ml, P<0.01, FIG. 1). The lowest autoantibody level
found in a five-month-old sHEL/Ig.sup.HEL /hCD19.sup.+/+ mouse was
2,300 ng/ml. Therefore, the breakdown of tolerance in
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice had 100% penetrance by five
months of age.
Abrogation of peripheral tolerance in sHEL/Ig.sup.HEL /hCD19 mice.
Whether B-cell anergy could be surmounted in young mice that
overexpress CD19 was assessed by immunizing two-month-old mice with
HEL in CFA. Mice without detectable levels of spontaneous anti-HEL
antibodies were also injected with CFA alone to mimic a nonspecific
inflammatory stimulus. Immunization of sHEL/Ig.sup.HEL
/hCD19.sup.+/+ mice with HEL generated primary anti-HEL antibody
responses in some mice, and a mean secondary antibody response that
was two hundred-fold higher (P<0.05) than that of
sHEL/Ig.sup.HEL mice (FIG. 2A). A measurable antibody response was
only detected in sHEL/Ig.sup.HEL mice after secondary immunization
(FIG. 2A). A striking result was that the inflammation induced by
CFA alone induced sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice to produce
anti-HEL antibodies in response to endogenous sHEL autoantigen
(FIG. 2A). In this case, the mean secondary antibody response was 4
thousandfold higher than in sHEL/Ig.sup.HEL mice (P<0.05). In
fact, the anti-HEL antibody levels induced in some sHEL/Ig.sup.HEL
/hCD19.sup.+/+ mice were equivalent to those of Ig.sup.HEL mice
(FIG. 2B). Similar results were obtained with sHEL/Ig.sup.HEL
/hCD19.sup.+/- mice although anti-HEL autoantibody levels were
intermediate. In sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice that already
expressed detectable anti-HEL antibodies, autoantibody levels were
also dramatically augmented following CFA administration.
Therefore, inflammatory responses induced by the administration of
CFA revealed a breakdown in tolerance and resulted in autoantibody
production in anergic mice that overexpressed CD19.
Effects of CD19 overexpression on B-cell development. The effects
of CD19 overexpression on B-cell development was assessed to
elucidate the cellular basis for the breakdown of peripheral
tolerance in sHEL/Ig.sup.HEL mice. The breakdown in tolerance did
not result from relaxed negative selection, since the number of
mature IgM.sup.+ B220.sup.hi or HSA.sup.lo B220.sup.hi B cells in
the bone marrow, blood, and spleen of Ig.sup.HEL /hCD19.sup.+/+
mice was significantly reduced in the absence or presence of sHEL
(Table 1).
B cell development in Ig.sup.HEL and sHEL/Ig.sup.HEL mice that
overexpress CD19 was analyzed using flow cytometry. The results are
discussed herein below. Representative two-color immunofluorescence
staining of B cells from A) bone marrow, B) blood, C) spleens, and
D) peritoneum of littermate pairs was performed. B lymphocytes were
revealed by B220 or IgM expression. Quadrants delineated by squares
indicated the pre-B cell (B220.sup.lo IgM.sup.-), immature B cell
(B220.sup.lo IgM.sup.+) and mature B cell (B220.sup.hi IgM.sup.+)
compartments, with numbers representing the percentage of cells
within quadrants. The gates that defined mature B lymphocytes for
sHEL/Ig.sup.HEL mice were different from the gate used for IgHEL
mice since surface IgM levels are downregulated in sHEL/Ig.sup.HEL
mice. Spleen cells were also stained for B220 or IgM and
counterstained for sHEL binding or I-A expression.
Additional gates were used to determine the frequency of the
CD5.sup.+ B220.sup.+ population and CD5.sup.- B220.sup.+ population
of cells for Table 1. Populations of cells lacking surface antigen
expression were determined using unreactive monodonal antibodies as
controls. All samples were stained in parallel and analyzed
sequentially by flow cytometry with identical instrument settings.
Relative fluorescence intensity was shown on a four decade log
scale, with 50% log density contour levels. Horizontal dashed lines
in some histograms were provided for reference. Similar results
were obtained with at least five sets of mice. Equivalent results
were obtained by using anti-IgM.sup.a antibody instead of anti-IgM
antibody.
A similar decrease in the generation of mature B cells occurs,
presumably as a consequence of increased clonal elimination, in
wild-type mice that overexpress CD19. Zhou et al. (1994) Mol. Cell.
Biol. 14:3884-3894. However, since all B cells bear the same
receptor with the same affinity for antigen, the partial decrease
in generation of mature B cells in the bone marrow of Ig.sup.HEL
/hCD19.sup.+/+ mice and sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice
suggests that this developmental bottleneck occurs independent of
antigen receptor ligation. Further, it is difficult to imagine a
deleting antigen that binds to the transgenic receptor better than
HEL, and deletion also occurs in mice lacking sHEL. Therefore,
overexpression of CD19 may alter the generation of mature B cells
through mechanisms in addition to increased negative selection.
Nonetheless, the breakdown in tolerance did not result from relaxed
negative selection.
Peripheral B cell numbers were significantly reduced in both
Ig.sup.HEL mice and sHEL/Ig.sup.HEL mice overexpressing CD19 (Table
1). Overexpression of CD19 reduced circulating B cell numbers by
87% in Ig.sup.HEL mice and 78% in sHEL/Ig.sup.HEL mice. CD19
overexpression reduced spleen B cell numbers by 42% in Ig.sup.HEL
mice and 48% in sHEL/Ig.sup.HEL mice. Conventional B cells within
the peritoneum were also reduced by >90% in Ig.sup.HEL
/hCD19.sup.+/+ and sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice.
Overexpression of CD19 did not induce the generation of B cells
with the phenotypic characteristics of either B1a or B1b cells and
only small numbers of CD5.sup.+ B220.sup.lo B cells were observed
in any of the 2-month-old transgenic mouse lines (Table 1). In
addition, all of the HEL-binding B cells in each line of mice were
conventional B cells since they were CD5.sup.-, CD23.sup.+,
IgD.sup.hi, and B220.sup.hi. Thus, the dramatic increase in the
levels of autoantibodies generated in sHEL/Ig.sup.HEL
/hCD19.sup.+/+ -transgenic mice were even more significant given
the >50% reduction in numbers of peripheral B cells in these
mice.
Chronic stimulation through the B-cell antigen receptor in
sHEL/Ig.sup.HEL mice results in a unique IgM.sup.lo IgD.sup.hi
phenotype with increased expression of class II (I-A) antigens
(Goodnow et al. (1988) Nature 334:676-682; Goodnow et al. (1989)
Nature 342:385-391; Mason et al. (1992) Intl. Immunol. 4:163-175).
In comparison, the overexpression of hCD19.sup.+/+ in these mice
resulted in even lower IgM expression and higher I-A expression
(Table 1). Despite the decrease in surface IgM, all B cells from
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice still bound sHEL in vitro in
proportion to their IgM.sup.a density. B cells from Ig.sup.HEL
/hCD19.sup.+/+ transgenic mice had an intermediate gM.sup.lo
I-A.sup.hi phenotype even in the absence of sHEL (Table 1). B cells
from mice that overexpressed CD19 also expressed significantly
elevated levels of cell surface CD86 (B7-2). Therefore, CD19
overexpression appeared to augment the phenotypic outcome of
signaling through the B cell antigen receptor in the absence or
presence of autoantigen. However, B cells from sHEL/Ig.sup.HEL
/hCD19.sup.+/+ mice still exhibited a phenotype that is
characteristic of anergic B cells.
[Ca.sup.++ ].sub.i responses in B cells from sHEL/Ig.sup.HEL /hCD19
mice. Peripheral tolerance in sHEL/Ig.sup.HEL mice results in the
failure of anergic B cells to mobilize intracellular Ca.sup.++ in
response to HEL-mediated antigen receptor crosslinking in vitro.
Cooke et al. (1994) J. Exp. Med. 179:425-438. B cells from
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice were equivalent to anergic B
cells from sHEL/Ig.sup.HEL mice in their failure to mobilize
Ca.sup.++ in response to HEL (FIG. 3A). B cells from
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice that generated high levels of
autoantibodies also failed to mobilize Ca.sup.++ in response to
HEL. B cells from Ig.sup.HEL /hCD19.sup.++ mice generated normal
Ca.sup.++ responses (FIG. 3B). Therefore, the development of
autoimmunity in sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice does not result
from a CD19-induced recovery of early signaling responses in the
bulk of anergic B cells.
Although antigen receptor ligation did not induce Ca.sup.++
responses in anergic B cells, crosslinking human and mouse CD19
induced a normal C.sup.++ response in anergic B cells from
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice (FIG. 3A). Crosslinking mouse
CD19 in sHEL/Ig.sup.HEL mice also induced a nominal Ca.sup.++
response. The presence of HEL during CD19 crosslinking resulted in
a Ca.sup.++ response that was significantly greater than that
observed with CD19 crosslinking alone in sHEL/Ig.sup.HEL
/hCD19.sup.+/+ mice (P<0.01, FIG. 3A). However, the magnitude of
the CD19/HEL-induced Ca.sup.++ response in sHEL/Ig.sup.HEL
/hCD19.sup.+/+ mice (FIG. 4A) was less than that observed in
Ig.sup.HEL /hCD19.sup.+/+ mice (FIG. 3B). Of interest was the
observation that the magnitude of the CD19-induced Ca.sup.++
response in sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice was always
significantly higher than the Ca.sup.++ response in Ig.sup.HEL
/hCD19.sup.+/+ mice (n=3, p<0.05). The increased Ca.sup.++
responses of B cells from sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice
presumably results from the endogenous ligation of antigen
receptors by sHEL encountered in vivo. These results indicate that
CD19 ligation can induce a relatively normal Ca.sup.++ response in
anergic B cells. Moreover, CD19 ligation can also augment
transmembrane signals generated through the B-cell antigen receptor
despite clonal anergy.
Thus, the striking induction of autoantibody production in
sHEL/Ig.sup.HEL mice that are normally functionally anergic
directly implicates CD19 signaling thresholds as a regulator of
peripheral tolerance in B cells. CD19 overexpression by only
twofold to threefold caused a breakdown of peripheral B-cell
tolerance in a clear and dramatic fashion, with autoantibody levels
increased several thousandfold in some sHEL/Ig.sup.HEL
/CD19.sup.+/+ mice (FIG. 1 and 2). These dramatic increases in
autoantibody levels in sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice are even
more significant given the >50% reduction in numbers of
peripheral B cells in mice that overexpress CD19 (Table 1).
Overexpression of CD19 alone did not induce anergic B cells to
produce autoantibodies, as evidenced by the fact that some
sHEL/Ig.sup.HEL /hCD19 mice were anergic and did not produce
spontaneous autoantibodies until 5 months of age (FIG. 1). However,
significant autoantibody production was induced in 2-month-old
anergic sHEL/Ig.sup.HEL /hCD19 mice by inducing inflammation with
CFA (FIG. 2). Autoantibodies in sHEL/Ig.sup.HEL mice that
overexpressed CD19 are likely to have originated from a breakdown
in tolerance in conventional B cells, since HEL-specific B1a or B1b
cells were not detected in Ig.sup.HEL (Cyster et al. (1995)
Immunity 2:13-24), sHEL/Ig.sup.HEL or sHEL/Ig.sup.HEL /hCD19 mice
(Table 1). These findings suggest that alterations in CD19-related
signaling thresholds breaks peripheral tolerance, which predisposes
B cells to the induction of autoantibodies.
The levels of autoantibody production observed in sHEL/Ig.sup.HEL
mice that overexpress CD19 (FIG. 1 and 2) clearly demonstrates that
tolerance was abrogated in a significant portion of B cells. Since
Ig.sup.HEL B cells are constantly exposed to antigen in transgenic
sHEL mice, autoantibody production in sHEL/Ig.sup.HEL /CD19.sup.+/+
mice is most likely induced through an antigen receptor-dependent
process. Autoantibody production in sHEL/Ig.sup.HEL /CD19.sup.+/+
mice may relate to the observation that CD19 ligation can augment
transmembrane signals generated through the antigen receptor
despite clonal anergy (FIG. 3).
Applicant has recently demonstrated that genetic alterations in
CD19 expression have significant effects on the signal transduction
pathways activated following B cell antigen receptor engagement.
Particularly, it was shown that CD19 and CD22 reciprocally regulate
Vav tyrosine phosphorylation during B lymphocyte signaling.
Therefore, one pathway to autoantibody production in
sHEL/Ig.sup.HEL mice may be via concomitant CD19 overexpression,
chronic antigen receptor ligation, and the influence of
inflammatory mediators triggering the simultaneous breakdown of
tolerance and autoantibody production in anergic Ig.sup.HEL B
cells.
Alternatively, inflammatory mediators such as those generated by
CFA administration may induce the expansion or differentiation of
antigen-stimulated Ig.sup.HEL B cell clones subsequent to a
CD19-induced breakdown in tolerance. The latter possibility is
supported by the finding that B cells from mice that overexpressed
CD19 maintained a phenotype characteristic of anergic B cells and
failed to generate Ca.sup.++ responses following antigen receptor
ligation (FIG. 3). The spontaneous development of autoantibodies in
sHEL/Ig.sup.HEL /hCD19 mice may also require a breakdown in T-cell
tolerance, since HEL-specific helper T cells are anergic due to
chronic sHEL exposure. Adelstein et al. (1991) Science
251:1223-1225. Thereby, soluble factors induced by CFA
administration may replace the requirement for T-cell help during
autoantibody production in sHEL/Ig.sup.HEL mice. Thus, the current
results suggest strongly that inappropriate CD19 expression or
function contributes to autoimmunity by disrupting tolerance.
Variability in the timing and magnitude of autoantibody production
in individual sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice is similar to
what has been observed in many mouse models of autoimmunity.
Theofilopoulos et al., eds. (1992) Murine models of lupus. Systemic
Lupus Erythematosus. Churchill Livingston, Edinburgh.
Overexpression of CD19 in sHEL/Ig.sup.HEL mice resulted in
significant autoantibody production in a large portion of
2-month-old mice, while all triple-transgenic mice produced
significant autoantibodies by 5 months of age (FIG. 1). The
expansion and/or accumulation of B-cell clones that have escaped
tolerance may explain why all sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice
produced high levels of spontaneous autoantibodies by 5 months of
age. This contrasts markedly with old sHEL/Ig.sup.HEL mice which
did not produce significant levels of anti-HEL autoantibodies (FIG.
1).
Previous studies of sHEL/Ig.sup.HEL mice have demonstrated that
functional inactivation of autoreactive B cells is maintained
throughout life. Rathmell et al. (1994) J. Immunol. 153:2831-2842.
The variability and delayed onset of autoantibody production in
sHEL/Ig.sup.HEL /hCD19.sup.+/+ mice may also result from their
confinement to a specific-pathogen-free barrier facility.
Autoantibodies first appear in some mouse models of lupus (NZB and
MRL strains) around 2 months of age, but are dramatically increased
by 4-5 months of age in all mice. Variability in onset and
magnitude of autoantibody production also occurs in individual mice
of these inbred mouse strains. Therefore, variability between the
triple transgenic mice examined in this study is not surprising
despite their identical genetic background and the fact that the
hCD19 transgene is expressed to the same extent in all animals.
Consistent with the current studies, an association between CD19
overexpression and autoimmunity in humans has been suggested. Oooto
et al. (1995) Jpn. J. Clin. Pathol. 43:381-384. The etiology of
autoimmunity in humans has also been historically linked with the
accumulation of inflammatory episodes or infectious agents and
often varies in degree and time of onset. Therefore, many of the
current findings in sHEL/Ig.sup.HEL mice mimic the evolution of
autoreactive B cells and autoantibodies in humans.
In contrast with CD19 overexpressing B cells, signaling in response
to antigen receptor ligation is diminished in CD45-deficient
Ig.sup.HEL B cells. Cyster et al. (1996) Nature 381:325-328.
Diminished signaling in CD45-deficient B cells leads to reduced
negative selection in the bone marrow and prolonged retention in
peripheral lymphoid tissues of mice expressing sHEL. Since the in
vivo functional capacity of peripheral CD45-deficient Ig.sup.HEL B
cells or their production of autoantibodies has not been examined,
it is difficult to assess how diminished signaling in those studies
relates to the results of this Example. Nonetheless, all of these
studies demonstrate that antigen receptor signaling strength
influences positive or negative selection, and the current studies
demonstrate a direct and active role for CD19 in regulating
peripheral tolerance and autoantibody generation.
For several reasons, it is unlikely that the breakdown in tolerance
observed in this study results from the inadvertent insertion of
the CD19 transgene into a locus that controls B cell tolerance.
First, the human CD19 transgenic mice used in these studies
reconstitute normal B cell function when crossed with
CD19-deficient mice. Sato et al. (1997) J. Immunol. 158:4662-4669.
The h19-1 line of mice has also been backcrossed extensively onto a
wild-type C57BL/6 background without a diminution of human CD19
expression. This suggests that only one transgene integration site
exists and that the heterogeneity in autoantibody production
observed between triple transgenic mice does not reflect the
segregated inheritance of transgenes that have integrated into
multiple sites.
Applicant also has generated and analyzed 7 independent lines of
hCD19 transgenic mice. Zhou et al. (1994) Mol. Cell. Biol.
14:3884-3894. In all cases, B cells from each mouse line
demonstrate identical functional abnormalities. In lines subjected
to analysis, hyper-responsive B cells and enhanced autoantibody
production was observed. The magnitude of these abnormalities
correlates directly and linearly with the level of hCD19
overexpression. Sato et al. (1997) J. Immunol. 158:4662-4669. In
addition, the abnormalities observed in hCD19 transgenic mice are
reciprocal of what applicant has observed in CD19-deficient mice
(Engel et al. (1995) Immunity 3:39-50; Sato et al. (1995) Proc.
Natl. Acad. Sci. USA 92:11558-11562; Sato et al. (1996) J. Immunol.
156:4371-4378). Therefore, the effects observed in the current
study most likely relate directly to augmented CD19 function rather
than an interruption of other genes involved in tolerance
regulation.
CD19 is a signaling component of a multimeric complex that includes
CD21, the receptor for the C3d fragment of complement that
covalently associates with antigens during complement activation.
Bradbury et al. (1992) J. Immunol. 149:2841-2850; Matsumoto et al.
(1991) J. Exp. Med. 173:55-64. C3d binding to CD21 can thereby act
as a ligand for the CD19 complex that links complement activation
with B-cell function. Pepys, M. B. (1976) Transplant. Rev.
32:93-120; Melchers et al. (1985) Nature 317:264-267; Fearon et al.
(1995) Annu. Rev. Immunol. 13:127-149.
Since CD21-deficient mice manifest developmental and functional
defects similar to those of CD19-deficient mice (Engel et al.
(1995) Immunity 3:39-50; Rickert et al. (1995) Nature 376:352-355;
Ahearn et al. (1996) Immunity 4:251-262; Molina et al. (1996) Proc.
Natl. Acad. Sci. USA 93:3357-3361) overexpression of CD19 in vivo
may mimic C3d ligation of CD21 by augmented signaling through the
CD19 complex. Tedder et al. (1997) Immunity 6:107-118. Because the
roadblock to B-cell Ca.sup.++ responses in anergic B cells was
transcended in vitro by simultaneous CD19 ligation and antigen
receptor signaling (FIG. 4), C3 cleavage products binding to the
CD19 complex may provide a molecular mechanism for bypassing
peripheral B-cell anergy in vivo. The inappropriate or prolonged
generation of C3d during inflammatory or infectious episodes in
vivo may increase the responsiveness of autoreactive B cells to
weak self antigens through augmented CD19 function, resulting in a
breakdown of tolerance and the clonal amplification of
autoantibody-producing B cells. Because altering CD19 complex
function provides a mechanism for breaking self-tolerance in vivo,
CD19 function may be a molecular mechanism linking inflammation
with the development of autoimmune disease.
TABLE 1 Phenotype and Frequency of B lymphocytes in Lymphoid
Tissues sHEL Ig.sup.HEL sHEL Ig.sup.HEL Ig.sup.HEL hCD19.sup.+/+
Ig.sup.HEL hCD19.sup.+/+ Tissue Phenotype Frequency (%) and Number
# .times. 10.sup.-6) of B cells* bone % IgM.sup.- B220.sup.lo 14
.+-. 2 14 .+-. 2 13 .+-. 2 13 .+-. 2 marrow % IgM.sup.+ B220.sup.lo
32 .+-. 4 49 .+-. 10 46 .+-. 2 53 .+-. 3 % IgM.sup.+ B220.sup.hi 16
.+-. 3 8 .+-. 3.sup..sctn. 12 .+-. 2 8 .+-. 1 % HSA.sup.lo
B220.sup.hi 25 .+-. 3 9 .+-. 3.sup.l 17 .+-. 5 5 .+-. 1.sup..sctn.
blood % B220.sup.+ 43 .+-. 3 7 .+-. 2.sup.l 22 .+-. 3 6 .+-.
2.sup.l # B220.sup.+ 2.4 .+-. 0.5 0.3 .+-. 0.1.sup.l 0.9 .+-. 0.2
0.2 .+-. 0.1.sup.l spleen % B220.sup.+ 36 .+-. 6 28 .+-. 5 44 .+-.
4 17 .+-. 3.sup.l # B220.sup.+ 24 .+-. 5 14 .+-. 3.sup..sctn. 29
.+-. 5 15 .+-. 6.sup..sctn. peritoneum % CD5.sup.+ B220.sup.lo 2.2
.+-. 0.3 1.3 .+-. 0.4 4.5 .+-. 1.1 1.9 .+-. 0.7 # CD5.sup.+
B220.sup.lo 0.05 .+-. 0.01 0.05 .+-. 0.02 0.10 .+-. 0.02 0.04 .+-.
0.02 % CD5.sup.- B220.sup.hi 38 .+-. 6 2.7 .+-. 0.7.sup.l 14 .+-. 2
1.2 .+-. 0.4.sup.l # CD5.sup.- B220.sup.hi 1.0 .+-. 0.3 0.07 .+-.
0.03.sup..sctn. 0.33 .+-. 0.03 0.02 .+-. 0.01.sup.l Expression
Source of B cells Levels Relative to Ig.sup.HEL Mice (% .+-.
SEM).sup..dagger-dbl. IgM levels bone B220.sup.lo 100 69 .+-.
12.sup.l 41 .+-. 9 22 .+-. 8 marrow: B220.sup.hi 100 63 .+-.
4.sup.l 4.1 .+-. 0.5 4.3 .+-. 0.7 blood: B220.sup.+ 100 68 .+-.
15.sup.l 19 .+-. 3 25 .+-. 4 spleen: B220.sup.+ 100 62 .+-. 4.sup.l
16 .+-. 2 7 .+-. 1.sup.l I-A levels: blood: IgM.sup.+ 100 126 .+-.
5.sup.l 169 .+-. 7 254 .+-. 33.sup.l spleen: IgM.sup.+ 100 259 .+-.
55.sup..sctn. 184 .+-. 13 283 .+-. 30.sup..sctn. * = Cumulative
mean (.+-. SEM) frequencies of different cell populations from at
least five two-month-old mice of each genotype. Flow cytometry
gates were used to determine the frequency of each cell type within
the lymphocyte population. B-cell numbers for blood indicate the
number of cells/ml. B-cell numbers from spleen and peritoneum were
determined based on the total number of lymphocytes recovered.
.sup..dagger-dbl. = Relative cell surface antigen densities were
determined by comparing the channel numbers of mean linear
fluorescence intensity between Ig.sup.HEL B cells and B cells from
other mice. Values represent the mean expression levels obtained
from at least three sets of mice of each genotype. All samples in
each set of mice were stained in parallel and analyzed sequentially
by flow cytometry with identical instrument settings. .sup.517 =
Differences between mice not expressing hCD19 and those expressing
hCD19 were significant, p < 0.05; .sup.l = p < 0.01.
EXAMPLE 2
Preparation of Monoclonal Antibodies against Prion Epitopes
As discussed above, monoclonal antibodies (mAbs) have become
powerful tools in research and biotechnology. In these areas, as in
the acquisition of immunity, production of potentially useful
antibodies often depends on two aspects of "self"-"non-self"
discrimination during the antibody response in mice. First,
antibodies are not normally produced against self antigens, and
secondly, antibodies to foreign antigens are normally directed
exclusively at regions of the foreign antigen that differ from
self. The number of monoclonal antibodies generated against
species-specific antigens for use in sensitive immunoassays or
allele-specific antibodies for blood grouping and tissue typing
before transfusion or organ transplantation has been quite
significant. However, the true potential of monoclonal antibodies
has not been realized in many cases because of the difficulty in
generating antibodies directed to self antigens or antigens well
conserved across species barriers.
This problem has resulted in the development of a number of
alternative approaches to generating monoclonal antibodies such as
phage display and other in vitro approaches. While there is
considerable power to these alternative approaches, the generation
of dramatic antibody diversity and clonal selection of high
affinity antibodies that occur normally during an immune response
in animals are impossible to recapitulate in vitro. Therefore,
effective methods, such as those described in Example 1, have been
developed wherein clonal tolerance is blocked in vivo and wherein
"self"-"non-self" discrimination is negated during the antibody
response in mice. Being able to modulate the molecular basis for
the cellular decision of tolerance is important for controlling the
immunogenicity or tolerogencity of vaccines, tumors, and tissue
transplants, and for understanding the breakdown of self-tolerance
in autoimmune diseases.
A number of molecules are key to regulating B cell function and the
generation of humoral immune responses. Two examples of such
molecules are CD19 and CD22. Tedder et al. (1997) Immunity
6:107-118; Tedder et al. (1997) Ann. Rev. Immunol. 15:481-504.
Recent studies have demonstrated that CD19 and CD22 are members of
a new class of lymphocyte surface receptors called "response
regulators". This term designates that these molecules regulate the
magnitude and duration of transmembrane signals received by a B
lymphocyte.
Understanding how these molecules function provides multiple
options for use in regulating humoral immune responses.
Specifically, mice have been generated that are hyporesponsive to
transmembrane signals, while other mice have been generated that
are hyper-responsive to transmembrane signals. This has
considerable ramifications for the abilities of these mice to
recognize and generate humoral antibody responses against a variety
of antigens, particularly self antigens. Moreover, this provides a
powerful new approach to developing a new class of mAbs that react
with molecules which are highly conserved during recent mammalian
evolution.
Unconventional agents termed prion proteins (PrPsc) are considered
the etiologic agent of Creutzfeldt-Jakob disease (CJD). The
pathologic properties of these proteins lie in their
three-dimensional configuration and their ability to recruit and
influence normal PrPs to undergo similar conformational changes.
Because these proteins are highly conserved across species it has
been difficult to generate effective humoral immune responses
against these agents in animal models.
The majority of PrPsc antigenic sites are species-directed, involve
non-self sites and are common to both the normal host precursor
(PrPc) and the disease form (PrPsc). Because of this, the present
methods to diagnose CJD-associated diseases are lengthy, require
relatively large quantities of starting material to detect PrPsc
and lack sensitivity (reviewed in Kascsak et al. (1993) Dev. Biol.
Stand. 80:141-151). See also Korth et al. (1997)(Letter to Nature)
Nature 390:74.
In this Example, development of monoclonal antibodies (mAbs)
reactive with PrPs is described. Development of systems for the
production of mAbs reactive with distinct epitopes or
conformational determinants present on PrPs but not PrPc are also
described.
Determination of whether tolerance-deficient mice develop humoral
immune responses to PrPc. Initial studies are initiated to
determine whether tolerance-deficient mice are able to generate
humoral immune responses against hamster PrPc or recombinant PrPc
proteins and peptides. Mice are immunized intra-pertioneally (ip)
with the test proteins and both primary and secondary humoral
immune responses are assessed by ELISA. Multiple
tolerance-deficient mice strains are assessed and are compared with
wild-type mice of the same genetic background.
Next, different immunogens are evaluated for their ability to
generate anti-PrP antibody responses to assess whether any have
specific advantages. One suitable immunogen is described in Korth
et al. (1997) (Letter to Nature) Nature 390:74.
Different gene-targeted or transgenic mouse models for
tolerance-deficiency are then assessed to determine advantageous
humoral immune responses following immunization. Upon observation
of clear differences between lines of mice, then those mice with
optimal humoral responses are crossed to generate a line of mice
that are optimal for generating humoral responses to PrPs.
In a second wave of experiments, tolerance-deficient mice are
generated that possess different major histocompatability complex
(MHC) backgrounds. In many experimental models, differences in MHC
genes have a predetermining effect on the ability of mice to elicit
humoral immune responses. MHC differences may account for some of
the difficulties in generating humoral responses to PrP protein in
mice.
Currently, all tolerance-deficient mice have a C57BL/6 background.
These mice are bred into a BALB/c background for at least five
generations. Humoral immune responses in mice is assessed following
initial crosses to assess whether this has an advantageous
influence on humoral immune responses. Following these crosses,
these mice are used for the generation of mAbs.
Generate mAbs Reactive with PrPc. The mice generated above and
immunized with optimal antigens are used for the generation of mAbs
using standard approaches, such as those described above.
A problem has been encountered in the development of hybridomas
producing antibodies reactive with PrP protein in which the myeloma
fusion partner cells express endogenous PrP protein which leads to
the death of the hybridoma. This problem is circumvented either by
genetically engineering the fusion partner myeloma cell line or
introducing the appropriate genetic mutations in the mice used for
fusions. These technologies will be utilized when it appears that
difficulties in generating mAbs results from the above problem.
Generate mAbs with PrP0/0 tolerance-deficient mice. Mice rendered
deficient in PrPc by homologous recombination gene targeting are
obtained. PrP0/0 mice generate sera capable of specifically
precipitating in vitro synthesized human prion proteins, while
wild-type mice do not generate humoral immune responses. Drasemann
et al. (1996) J. Immunol. Methods 199:109-118). This provides
considerable advantages for generating mAbs reactive with this
self-protein should responses with tolerance-deficient mice not be
optimal. Furthermore, this may allow the generation of mAbs
reactive with novel epitopes that may not be optimally identified
in tolerance-deficient mice. PrP0/0 tolerance-deficient mice
generate vigorous polyclonal immune responses after immunization
with human prion gene sequences. These mice will be used as spleen
donors for mAb production.
Generate mAbs reactive with "infectious" PrP isoforms. For this
phase, steps are taken to adequately protect laboratory and animal
care personnel against potential infectious materials. Monoclonal
antibodies are then preferably generated by DNA-mediated
immunization of mice, or by other standard protocols known to those
in the art.
Additionally, high affinity monoclonal antibodies are prepared by
the methods described in this Example. Monoclonal antibodies having
high affinity for an antigen are desirable in that high affinity
provides for biological activity in vivo. Some monoclonal
antibodies produced by conventional methods often react with
antigens in a biological sample, such as blood, but the affinity of
such reactions is low.
The terms "affinity" and "high affinity" have well-recognized
meanings in the art. Particularly, the term "affinity" refers to
the goodness of the fit of an antigenic determinant to a single
antigen-binding site, and it is independent of the number of sites.
The term "high affinity" refers to a particular good fit of an
antigenic determinant to a single antigen-binding site.
Typically, the affinity of an antibody and an antigenic determinant
is judged by the use of an affinity constant K. Antigens (Ags) and
antibodies (Abs) interact according to the reversible equilibrium
equation:
The affinity constant K is determined by the equation
K=[AgAb]/[Ag][Ab], wherein the units for K are liters per mole.
The affinity constant for a particular antigen-antibody interaction
can be determined by measuring the concentration of free Ag
required to fill half the antigen binding sites on the antibody.
When this concentration value is plugged into the equation
presented above, K=1/[Ag]. Thus, the reciprocal of the antigen
concentration that produces half maximal binding is equal to the
affinity constant of the antibody for the antigen. A high affinity
antigen-antibody interaction is therefore typically described as
having an affinity constant K of greater that 1.times.10.sup.5
liters per mole. See Alberts et al. Molecular Biology of the Cell,
Garland Publishing, Inc. (New York and London. 1983), p. 970.
Such high affinity monoclonal antibodies have application, for
example, in a pharmaceutical setting. Specific binding of an
antigen, such as a cancer antigen, can prevent the antigen from
carrying out its biological activity in the cell with which it is
associated, thereby killing the cell or slowing its growth.
Alternatively, such monoclonal antibodies have application as drug
carriers, for delivery of, for example, a cytotoxic agent to the
cancer cell.
Production of monoclonal antibodies directed to cancer-associated
antigens are also described in U.S. Pat. Nos. 5,660,834; 5,491,088;
5,665,382; 5,651,991; and 5,242,824; the entire contents of which
is herein incorporated by reference.
EXAMPLE 3
Preparation of a Non-Human Animal having an Immune System with
Desired Characteristics
Example 1 describes breeding of transgenic mice, including a
transgenic mouse which over-expresses CD19, wherein progeny of such
mice possess antibody-forming cells having disrupted peripheral
tolerance. Given the disclosure of Example 1, then, a practitioner
having ordinary skill in the art can breed and/or prepare lines
transgenic mice for cross-breeding, wherein each line of mice have
immune systems with certain characteristics, to produce progeny
animals having combinations of the characteristics. Such animals
would be very useful, inter alia, in the preparation of monoclonal
antibodies as described herein or in the preparation of vaccines.
This Example describes the preparation of such an animal.
An example of a desirable characteristic is hyper-sensitivity to
antigens so that antibodies, and particularly monoclonal
antibodies, can be made against epitopes that are highly
conservative among mammalian or other vertebrate species. Indeed,
an animal having a hyper-sensitive immune system due to a breakdown
in peripheral tolerance is described in Example 1.
In producing a non-human animal having an immune system with a
predetermined or desired characteristic, it is preferable to
initially prepare a transgenic animal which includes a transgene,
wherein expression of the transgene in the animal imparts a
predetermined or desired characteristic to a cell or a type of
cells within the animal's immune system. As shown in Example 1,
transgenic mice which over-express CD19 are used to provide
disruption of peripheral tolerance in B lymphocytes in mice.
Preparation of transgenic animals is accomplished through use of
accepted protocols in the art. Such protocols are described, for
example, in Engel et al. (1995) Immunity 3:39-50 and in Zhou (1994)
Mol. Cell. Biol. 14:3884-3894, wherein the production of the CD19
overexpressing transgenic mice described in Example 1 is described.
Additionally, standard preparation techniques for transgenic
animals, including mice, are also described in U.S. Pat. No.
5,633,076 (bovine); U.S. Pat. No. 5,573,933 (pigs); U.S. Pat. No.
5,675,063 (rabbits); U.S. Pat. No. 5,633,425 (mouse); U.S. Pat. No.
5,661,016 (mice and other animals) and U.S. Pat. No. 4,736,866
(mice and other animals), the entire contents of each of which are
herein incorporated by reference.
The preferred next step in the method is to prepare a second line
of transgenic animals which include a transgene, wherein expression
of the transgene in the animal imparts a predetermined or desired
characteristic to a cell or a type of cells within the animal's
immune system. The predetermined characteristic of the cell or
cells within the immune system of the second strain of transgenic
animals can be the same or different from the predetermined
characteristic of the cell or cells within the immune system of the
first strain of animals. In Example 1, the second and subsequent
lines of animals included sHEL (ML5 line) and Ig.sup.HEL (ML5 line)
mice, as well as double transgenic sHEL/Ig.sup.HEL mice.
Next, the first and second strains of transgenic animals are bred
according to classical breeding techniques. Such techniques are
well known in the art, and are be more fully described in U.S. Pat.
No. 5,633,076 (bovine); U.S. Pat. No. 5,573,933 (pigs); U.S. Pat.
No. 5,675,063 (rabbits); U.S. Pat. No. 5,633,425 (mouse); U.S. Pat.
No. 5,661,016 (mice and other animals) and U.S. Pat. No. 4,736,866
(mice and other animals), the entire contents of each of which are
herein incorporated by reference.
Additionally, techniques for the care and breeding of a germ-free
colony of mice are described in U.S. Pat. No. 5,223,410, the entire
contents of which is herein incorporated by reference. Such
techniques may optionally be incorporated into the production of
the animal having an immune system with predetermined
characteristics as described in this Example.
The resulting progeny are then screened to determine that
transgenes are inherited and are subsequently expressed. Screening
protocols include the well known techniques of PCR amplification,
northern blot analysis and southern blot analysis using nucleic
acid probes or segments from the transgene initially used to
prepare the transgenic animal. Additional protocols are described
in Engel et al. (1995) Immunity 3:39-50 and in Zhou (1994) Mol.
Cell. Biol. 14:3884-3894.
Immune cells within the progeny are then screened to determine if
the predetermined characteristics have been imparted to the cells.
Such screening methods are provided in Example 1 and include
isotype-specific ELISA assays and immunofluorescence assays.
Finally, progeny having immune cell or cells, such as
antibody-producing cells like B lymphocytes, are bred according to
well-known techniques to propagate a line of mice which have within
their immune systems cells which demonstrate the predetermined or
desired characteristics.
In addition to the lines of mice described in of Example 1,
applicant has prepared a line of CD22 deficient mice and a line of
CD19 deficient mice according to standard "knock-out" methods, such
as those methods described in Sato et al. (1996) Immunity
5:551-562. The CD22 deficient line of mice exhibit a positive
effect on antigen recognition, while the CD19 deficient line of
mice exhibit a negative effect on antigen recognition. The lines of
mice were crossed. The resulting line of mice exhibited normalized
B cell development in contrast to the abnormal B cell development
observed in CD22 deficient and CD19 deficient mice.
As another example of a predetermined or desired characteristic to
be manipulated, the role of antigen receptor signaling strength in
the development of autoreactive B cells has recently been examined
in sHEL/Ig.sup.HEL mice. Cyster et al. (1995) Immunity 2:13-24;
Cyster et al. (1996) Nature (London) 381:325-328. Mutations in the
SHP1 protein tyrosine phosphatase of motheaten viable (me.sup.v)
mice abrogates the negative regulatory role of SHP1 in antigen
receptor signaling, resulting in the generation of autoantibodies
in non-transgenic mice. In Ig.sup.HEL mice, the me.sup.v mutation
lowers signaling thresholds, which incites the negative selection
of Ig.sup.HEL B cells in the bone marrow of sHEL mice. Cyster et
al. (1995) Immunity 2:13-24. The SHP1 deficiency thereby prevents
autoantibody generation but facilitates the development of
peritoneal B1 cells reactive with HEL. Cyster et al. (1995)
Immunity 2:13-24.
These characteristics contrast markedly with the results of Example
1, in which lowering B-cell signaling thresholds by increased CD19
expression resulted in a breakdown in tolerance and autoantibody
production rather than the total negative selection of Ig.sup.HEL B
cells in the bone marrow of sHEL mice (FIG. 1). Thus, SHP1 may play
a key role in setting thresholds for negative selection in the bone
marrow, while CD19 regulates peripheral tolerance. Alternately,
tolerance may be finely tuned, with CD19 and SHP1 altering
signaling strengths to differing extents. Thus, animals can be
produced according to the methods of this Example wherein the
animals have cells in their immune systems that display
characteristics associated with altered SHP1 and CD19
expression.
Other examples of predetermined or desired characteristics would be
apparent to one having ordinary skill in the art. See, for example,
a review article by Tedder et al., entitled "The CD19-CD21 Complex
Regulates Signal Transduction Thresholds Governing Humoral Immunity
and Autoimmunity", Immunity 6:107-118 (February 1997), which
discusses the role played by the CD19-CD21 complex in the immune
systems of vertebrates.
The CD19 molecule forms a complex with CD21 (also known in the art
as complement receptor Type II [CR2]), CD81 and Leu-13. Tedder et
al. (1994) Immunology Today 15:437-442. Additionally, the structure
and in vitro function of the CD19-CD21 complex has been
characterized in the art. Tedder et al. (1994), Immunol. Today
15:437-442. Summarily, CD19 is a member of the immunoglobulin
superfamily with a cytoplasmic region of approximately 240 amino
acids. The amino acid sequences of the cytoplasmic of human CD19
(hCD19), mouse CD19 (mCD19), and guinea pig CD19 are highly
homologous, which is consistent with a critical role for this
region in CD19 function. CD19 physically associates with CD21 on
the surface of human B cells. CD21 contains an extracellular domain
of 15 or 16 repeating structural elements called short consensus
repeats (SCRs), a membrane spanning region, and a 34-amino acid
cytoplasmic domain. Human and mouse forms have been identified. The
human form, hCD21, can physically associate with a structurally
similar complement receptor, CD35 (CR1) and generate a receptor
complex that does not contain CD19. The human CD35 is expressed by
B cells, erythrocytes, neutrophils, monocytes, and some T cells.
Thus, interactions of these molecules can be manipulated to prepare
a non-human animal having an immune system with predetermined or
desired characteristics associated with such a manipulation.
CD19 also associates directly with CD81, a member of the tetrospans
family of proteins that includes CD9, CD37, CD53, CD63 and CD82.
Bradberry et al. (1992), J. Immunol. 149:2841-2850 and Levy et al.
(1991) J. Biol. Chem. 266:14597-14602. CD81 is over-expressed by
most B lineage cells and by a wide variety of cell types including
most lymphocytes, natural killer cells, thymocytes, eosinophils,
neuroblastomas, melanomas, and fibroblasts. Thus, interactions of
CD19 and CD81 within the complex can be manipulated to prepare a
non-human animal having an immune system with predetermined or
desired characteristics associated with such a manipulation.
Further, Table 2 presents phenotypic characteristics of mice with
genetically altered response regulators of B lymphocyte signal
transduction. The table presents both negative and positive
effects, each such effect being a potential predetermined
characteristic for an animal prepared according to the methods of
this Example.
TABLE 2 Phenotypic Characteristics of Mice with Genetically Altered
Response Regulators of B Lymphocyte Signal Transduction Genotype
Conventional B Cells CD5+/B-1 Cells Negative Effects CD19 deficient
.dwnarw.50% decrease .dwnarw..dwnarw.80% decrease CD21 deficient
Normal .dwnarw.40% decrease BTK deficient .dwnarw.40% decrease
.dwnarw..dwnarw..dwnarw.99% decrease Xid .dwnarw..dwnarw.70%
decrease .dwnarw..dwnarw..dwnarw.99% decrease Vav deficient
.dwnarw..dwnarw..about.Normal Undetectable Positive Effects CD19
.dwnarw..dwnarw.70% decrease .uparw..uparw..uparw.210%
Overexpressed increase SHP1 defective .dwnarw..dwnarw..dwnarw.
.uparw..uparw..uparw. CD22 deficient .dwnarw.50% decrease in
.uparw.Increase circulating B cells Lyn deficient .dwnarw.50%
decrease Normal Arrows represent the relative effect of the genetic
alteration on B cell development
As shown in Table 2, additional examples include Btk-deficient mice
and Xid mice with mutations in Btk both have diminished numbers of
B-1 and conventional B cells. Vav-deficient mice have similar
defects.
In addition to the examples set forth in Table 2, it is also
optionally desirable to prepare an animal wherein the C3 or C3d
binding component to the CD19-CD21 complex is depleted. It is known
in the art that such a depletion leads to impaired humoral
responses to T-cell dependent and some T-cell independent antigens
in mice. Pepys (1974) J. Exp. Med., 140:126-145.
In vivo complement depletion suppresses the production of IgG and
other T-cell dependent antibody classes much more significantly
than T-cell independent IgM responses. Transient depletion of C3
also completely abrogates the development of memory B cells. Klaus
and Humphrey (1977) Immunology 33:31-40.
Complement deficiencies that effect C3 activation in humans, guinea
pigs, and dogs result in diminished humoral responses to foreign
antigens. See, for example, O'Neil et al., (1988) J. Immunol.
140:139-145. Additionally, C3- and C4-deficient mice suffer severe
defects in both their primary and secondary antibody responses to
T-cell dependent antigens, even at high antigen doses.
A novel C3 mRNA transcript has been identified that encodes a
truncated C3 protein. Cahen-Kramer et al. (1994), J. Exp. Med.
180:2079-2088. Cell lines transfected with the related cDNA secrete
a co-stimulatory factor that augments the proliferation of B cells
in assays with macrophage-depleted mouse splenic B cells. Such a
cDNA thus provides a candidate for use in the production of a
transgenic animal according to the methods of this Example.
Additionally, CD20 and CD35, or CD21/35 play a role in immune
response, and are thus candidates for manipulation in an animal
according to the method of this Example. It has been observed that
pre-treatment of mice with a mCD21/35 MAb blocks both T-cell
dependent and independent immune responses in the generation of
immunological memory. See, for example, Gustavsson et al. (1995) J.
Immunol. 154:6524-6528. Chimeric mice with normal levels of CD21/35
on their follicular dendritic cells, but not on their B cells, have
defects in humoral responses to antigens similar to those of
CD21/35 deficient mice. Croix et al. (1996) J. Exp. Med.
183:1857-1864.
Additional candidates of interest include CD5.sup.- B cells and
CD5.sup.+ B-1 cells, given their documented role in pathogenic
autoantibody responses, such as the pathogenic autoantibody
responses of systemic lupus erythematosus (SLE). SLE is
characterized by production of antibodies to DNA within the body in
association with systemic inflammation. Shirai et al. (1991) Clin.
Immunol. Immunopathol. 59:173-186. See also Murakami and Honjo
(1995), Immunol. Today 16:534-539, discussing that high affinity
pathogenic IgG antibodies are generally produced by CD5.sup.- B
cells.
In summary, multiple response regulators govern signaling
thresholds in the cells of an animal's immune system, particularly
B cells. Response regulators with positive or negative effects
influence signaling through the B cell antigen-receptor complex.
The resulting signaling thresholds regulate negative selection in
the bone marrow, the magnitude of antibody response in the
periphery, autoimmunity, and peripheral tolerance. Therefore,
preparing an animal having cells in its immune system with
predetermined characteristics derived from manipulation of these
response regulators is highly useful in the characterization of
immune response, development of monoclonal antibodies, and
vaccines, and in the treatment of autoimmune disorders.
Given that methods for the production of transgenic animals
well-known in the art, it is believed that any animal can be
utilized in the methods of this Example, including mouse, pig, rat,
rabbit, guinea pig, goat, sheep, primate, and poultry, with a mouse
being preferred. Additionally, by the term "transgenic", it is
meant any animal having a genome altered by the hand of man in any
manner. Thus, the term "transgenic" includes the insertion of
desired transgene, any of the well-known "knock-in" approaches, and
any of the well-known "knock-out" approaches.
Furthermore, the preparation of lines of animals having immune
system cells demonstrating a predetermined or desired
characteristic for use in subsequent breeding is not limited to
transgenic protocols. Any suitable protocol that generates an
alteration in the cells of the animal's is contemplated to be
within the scope of the method of this Example. Such protocols
include, among others, exposure to mutagens, as described in Cyster
et al. (1995) Immunity 2:13-24.
EXAMPLE 4
Alteration of Immune Response to NP Hapten in Mice
C57BL/6 mice generate T cell-dependent humoral responses to the
(4-hydroxy-3-nitrophenyl)acetyl (NP) hapten that are dominated by
canonical antibodies composed of a single V.sub.H gene, V186.2, and
.lambda.1 light chain. Selection for this receptor is thought to be
driven by its frequency and affinity. However, lowering the
activation threshold of B lymphocytes by overexpression of a single
cell-surface molecule, CD19, resulted in anti-NP antibodies
comprising an unprecedented diverse repertoire of V.sub.H and
V.sub.L rearrangements with no or few mutations. Remarkably, many
exhibited affinities for NP greater than or equal to that of
canonical antibodies. Thus, antigen-receptor selection is regulated
by endogenous B lymphocyte signaling thresholds and not antigen
receptor affinity.
The unprecedented diverse repertoire of V.sub.H and V.sub.L
rearrangements presented in this Example demonstrates the
flexibility and broad ranging applicability of methods of the
present invention. Indeed, the ability to alter cells of the immune
system in an animal is further exemplified by the detailed
characterization of the unprecedented immune response to NP
presented in this Example. The production of monoclonal antibodies
to highly conserved epitopes and the production of a non-human
animal having cells of the immune system with a predetermined
characteristic in accordance with the methods of the present
invention is thus further illustrated by the data presented in this
Example.
Background
Despite the enormous diversity of antibodies, inbred strains of
mice often respond to haptens and simple antigens by producing
remarkably homogenous antibodies (Blier and Bothwell, 1988). One of
the best examples is the response of C57BL/6 (Igh.sup.b) mice to
the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten (Imanishi and
Makela, 1975). C57BL/6 mice immunized with NP coupled to protein
carriers generate serum antibodies which bear the normally rare
.lambda.1 light chain (Cumano and Rajewsky, 1986; Imanishi and
Makela, 1975; Jacob et al., 1991; Karjalainen et al., 1980; Makela
and Karjalainen, 1977; Reth et al., 1978; Reth et al., 1979; Weiss
and Rajewsky, 1990). Immunization with carrier protein alone
elicits virtually no .lambda.1 antibody or A1.sup.+ B cells. Early
in the immune response (days 4-8 post-immunization) a large
proportion of activated .lambda.1.sup.+ B cells express multiple D
gene segments in combination with various members of the large J558
family of V.sub.H genes, including V186.2, C1H4, CH10, V23, 24.8,
V102, and V583.5 (Allen et al., 1988; Bothwell et al., 1981; Jacob
and Kelsoe, 1992; Jacob et al., 1993). By day 10 after
immunization, the majority of .lambda.1.sup.+ B cells express
V186.2-to-DFL16.1 gene rearrangements that encode a tyrosine-rich
CDR3 region with a consensus motif, YYGS (Bothwell et al., 1981;
Cumano and Rajewsky, 1985; Jacob et al., 1993; McHeyzer-Williams et
al., 1993; Weiss and Rajewsky, 1990). The V186.2-to-DFL16.1 heavy
chain rearrangement paired with the .lambda.1 light chain is
referred to as the canonical anti-NP B cell antigen receptor (Reth
et al., 1978; Reth et al., 1979).
The homogeneity of the anti-NP response in Igh.sup.b mice (Maizels
and Bothwell, 1985) is mirrored in the response of BALB/c mice to
phosphorylcholine (Crews et al., 1981); antibodies produced against
p-azophenylarsonate in strain A mice (Pawlak et al., 1973); the
2-phenyloxazolone response in BALB/c and DBA/2 mice (Makela et al.,
1978); and the response of BALB/c mice to poly(Glu.sup.60
-Ala.sup.30 -Tyr.sup.10) (Theze and Somme, 1979). The cause of low
genetic variance in these antibody responses remains obscure.
Linkage of restricted antibody responses to single VDJ gene
segments or Igh alleles suggests an occasional, single best
solution to antigen-complementarity that results in expansion of
that B cell lineage. In this case, strain-specific differences in
the repertoire of germline V.sub.H genes would regulate the
antibody response. Alternatively, several investigators have
suggested that restricted antibody responses are circumscribed by
self-tolerance; others note that clones expressing V(D)J
rearrangements that are robustly tolerant of mutational change
should outgrow mutationally fragile competitors. Nonetheless, the
mechanisms driving repertoire selection has remained a
controversial issue. Together, these observations have suggested
that the great specificity of humoral immune responses is not the
consequence of highly selective clonal activation but of
competitive survival and proliferation of higher affinity B
cells.
Transmembrane signals generated through the B cell antigen receptor
complex regulate B cell responses to antigen binding and may
thereby also regulate repertoire selection. Other cell surface
molecules can also modifying B cell responses to antigen.
Transmembrane signals generated through the B cell antigen receptor
complex and other surface receptors are critically regulated by
CD19 expression (reviewed in Fearon and Locksley, 1996; Tedder et
al., 1997). CD19 functions as a general regulator of B cell
proliferation, differentiation, clonal expansion in the peripheral
B cell pool, and of peripheral tolerance, as described above.
Whether differences in endogenous signal transduction thresholds
influence repertoire selection was assessed in this Example using
C57BL/6 mice with single complementary genetic alterations that
result in either the loss or overexpression of the CD19 cell
surface molecule. In general, B lymphocytes from CD19-deficient
(CD19-KO) mice are hypo-responsive to transmembrane signals while B
lymphocytes from transgenic mice that overexpress CD19 (CD19-TG)
are hyper-responsive (Engel et al., 1995; Sato et al., 1995; Zhou
et al., 1994). Indeed, transgenic mice with even small increases
(10-25%) in CD19 receptor density on B cells exhibit quantitative
changes in B cell functional capacity. Thus, CD19-deficient and
CD19-overexpressing mice serve as model systems where CD19 is a
general response regulator of cell-surface receptor signaling and
cellular signal transduction thresholds.
To assess the contribution of antigen receptor signaling to
repertoire selection, CD19-TG, CD19KO, and wildtype C57BL/6 mice
were immunized with NP coupled to the T-cell-dependent antigen,
chicken gamma globulin (CGG). Mice that overexpressed CD19
generated anti-NP humoral immune responses that were quantitatively
similar to those of wildtype C57BL/6 mice, while CD19KO mice
generated only modest responses. However, the antibody response
generated by mice that overexpressed CD19 were qualitatively
distinct from those of C57BL/6 controls. V.sub.H gene segment and
gene family use were unprecedently diverse in CD19TG mice and none
of the anti-NP antibodies produced carried .lambda.1 light chains.
Significantly, some of the atypical antibodies bound the NP hapten
better than canonical antibodies predominantly generated in
wild-type C57BL/6 mice. In addition, several of the anti-NP
antibodies generated by CD19-TG mice were reactive with a self
antigens. Thus, endogenous signaling thresholds regulate repertoire
diversity and selection during B cell responses to antigen
independently of change in the immunoglobulin loci.
Anti-NP Immune Responses in Mice Overexpressing CD19
CD19TG and wildtype C57BL/6 mice were immunized with NP.sub.18 -CGG
to assess their humoral immune responses to NP. Serum anti-NP
antibody concentrations at the time of immunization (day 0) and on
subsequent days (4, 8, 10, 16, and 58) were determined by ELISA
using NP.sub.25 -BSA. Values represented either mean antibody
levels (.+-.SEM) from 4-5 individual mice per time-point following
immunization relative to isotype-matched anti-NP monoclonal
antibodies used to generate standard curves, or represented
antibody concentrations relative to control serum from a C57BL/6
mouse immunized with NP.sub.18 -CGG. CD19TG mice generated serum
IgM responses quantitatively similar to those of wildtype mice,
despite an overall (.about.80%) reduction in peripheral B cell
numbers (Engel et al., 1995). IgG1 responses of CD19TG mice were
.about.10-fold lower than in wildtype mice. IgG2a, IgG2b, and IgG3
responses in CD19TG mice were also below those of wildtype mice.
All antibody responses were of the Igh.sup.b allotype. Remarkably,
.lambda.1 antibody responses were poor in CD19TG mice, while
.kappa..sup.+ antibody responses were similar in CD19TG and
wildtype mice. Therefore, the primary NP-specific antibody response
in CD19TG mice is dominated by IgM and IgG1, with the vast majority
of antibodies bearing light chains other than .lambda.1.
The relative affinity/avidity of the antibody response in CD19TG
mice was assessed by comparing antisera binding to
highly-(NP.sub.25 -BSA) or sparsely-(NP.sub.5 -BSA) substituted
NP-bovine serum albumin (BSA) substrates over a wide range of
antibody concentrations as described previously (Herzenberg et al.,
1980). Values represented mean (.+-.SEM) ratios of anti-NP.sub.5
versus anti-NP.sub.25 antibody concentrations from 4-5 individual
mice per time-point. In cases where anti-NP5 antibody levels were
not detectable, values of 0 were used for generating means.
Differences between CD19TG and wildtype mice were significant,
p<0.05, at about 3 days, 10 days and 14 days after immunization
for IgM; at about 11 days, 18 days and 58 days for .lambda.1; and
at about 58 days for .kappa..
Despite similar levels of IgM anti-NP antibodies, the primary
antibody response in CD19TG mice was generally of a lower affinity
compared with antiserum from wildtype mice. However, the IgG1
antibody responses in CD19TG and wildtype mice exhibited comparable
affinity maturation. Again however, the .lambda.1 antibody response
was modest and this response was of lower affinity than observed in
wildtype mice. It therefore appears that in response to NP, mice
overexpressing CD19 generate high affinity IgG1 antibody that does
not bear .lambda.1 light chain.
The relative frequency of anti-NP antibody-producing B cells in the
spleen and bone marrow of CD19TG mice was assessed using ELISpot
assays. Determined values represented mean AFC numbers (.+-.SEM)
from 4-14 individual mice per time-point following immunization on
day 0. Differences between CD19TG and wildtype mice were
significant, p<0.05, at about days 8 and 11 for IgM in spleen,
and at about day 58 for IgG1 in bone marrow.
NP-specific IgM antibody producing cells were 2- to 7-fold higher
among CD19TG mouse splenocytes than among wildtype splenocytes
following immunization. The frequency of IgG1 anti-NP
antibody-forming cells among CD19TG splenocytes were not
significantly different from wildtype controls. CD19TG mice also
had consistently higher frequencies of IgM-secreting cells and
lower frequencies of IgG-secreting cells among bone marrow-derived
anti-NP antibody-forming cells when compared with wildtype
controls. Importantly, the overall kinetics of NP-specific
antibody-forming cell responses were similar in CD19TG and wildtype
mice, although the long-lived memory antibody-forming cells
normally found in the marrow of wildtype mice 58 days after
immunization were not present in CD19TG mice. Therefore, the serum
antibody responses observed in CD19TG and wildtype mice generally
mirrored the antibody-forming cell responses.
Anti-NP Immune Responses in CD19KO Mice
CD19KO mice were immunized with NP.sub.18 -CGG to compare their
humoral immune responses with wildtype littermates. Mean serum
anti-NP antibody concentrations (.+-.SEM) from 4-5 individual mice
per time-point following immunization (day 0) were determined by
ELISA using NP.sub.25 -BSA as described above. The average relative
affinity of serum anti-NP antibodies was estimated by determining
the mean concentration of NP.sub.5 -binding and NP.sub.25 -binding
antibodies at each time-point by ELISA as described above.
Differences between CD19KO and wildtype were significant,
p<0.05, at about 3 days and 18 days after immunization for IgM;
at about 18 days and 58 days for IgG1; at about 58 days for
.lambda.1; and at about 18 and 58 days for .kappa..
The frequency of spleen and bone marrow cells secreting
anti-NP.sub.25 -binding antibody was determined by ELISpot assays
as described above. Values represent mean antibody-forming cell
(AFC) numbers (.+-.SEM) from 4-5 individual mice per time-point
following immunization on day 0. Differences between CD19KO and
wildtype mice were significant, p<0.05, at about days 10 and 18
for IgG1 in spleen, and at about days 10, 16 and 58 for IgG1 in
bone marrow.
Serum IgM and IgG1 antibody responses to NP were 10-fold and
>100-fold lower in CD19KO mice than in wildtype mice. .lambda.1-
and .kappa.-bearing antibody responses to NP were also suppressed
in CD19KO mice. Affinity maturation was also delayed and reduced in
CD19KO mice when compared with wildtype mice. The relative
frequency of B cells producing IgG1 anti-NP antibodies in the
spleens and bone marrow of CD19KO mice was markedly lower than in
wildtype mice at each time point following immunization. Therefore,
antibody responses to NP in CD19KO mice were markedly diminished
beyond what would be expected with the overall (40-60%) reduction
in peripheral B cell numbers in these mice.
Germinal Center Responses in CD19TG and CD19KO Mice
Germinal center B cell responses in CD19TG and CD19KO mice were
assessed after immunization with NP.sub.18 -CGG. Histologic
sections of spleen were stained with the GL7 monoclonal antibody
and/or peanut agglutinin (PNA) to identify germinal center B cells
(Laszlo et al., 1993; Rose et al., 1980). Antibody specific for
mouse .kappa. light chain was used to visualize the B cell zones
(follicles) within the splenic white pulp. Overall, B cell
follicles were smaller in CD19TG mice than in wildtype mice both
before and after NP immunization. Correspondingly, T cell zones
occupied a larger portion of CD19TG mouse spleens. The overall
frequency of follicles was also significantly (p<0.01) lower in
CD19TG mice than in wildtype mice, reflecting reduced B cell
numbers in these mice.
The frequency of PNA.sup.+ germinal centers within follicles of
CD19TG mice increased in response to immunization, although the
germinal centers were usually smaller in CD19TG mice than in
wildtype controls. The frequency of germinal centers per follicle
was also significantly reduced in CD19TG mice. Nonetheless, the
percentage of splenic B cells induced to express the GL7 antigen
was similar in CD19TG and wildtype mice as assessed by flow
cytometry, although GL7 expression kinetics were delayed in CD19TG
mice. However, the increase in GL7.sup.+ B cells in CD19TG mice at
day 16 was due to a significant number of GL7.sup.+ B cells found
outside of PNA.sup.+ germinal centers, something not observed in
C57BL/6 mice. In fact, there were relatively few GL7.sup.+ B cells
within the germinal centers of CD19TG mice on day 16 but there were
significant numbers of GL7.sup.+ cells with abundant cytoplasmic
immunoglobulin clustered around the penicilliary arterioles. Thus,
germinal centers form following antigen challenge of CD19TG mice
but they are small in size and dissipate at a faster rate than in
wildtype mice. Moreover, the germinal centers of CD19TG mice
displayed different phenotypic properties than observed in wildtype
mice by the dissociation of the PNA and GL7 markers. In further
contrast with results obtained in normal mice, germinal centers in
CD19TG mice did not contain .lambda.1.sup.+ B cells after
immunization with NP-CGG.
The number and size of primary follicles in CD19KO mice were
normal, but generated few germinal centers or GL7.sup.+ B cells
following NP-immunization. Similarly, .lambda.1.sup.+ B cells were
not observed in the few germinal centers that formed in CD19KO mice
after immunization with NP. Therefore, NP-immunization did not
induce a significant germinal center response in CD19KO mice.
Anti-NP Antibody Diversity in CD19TG Mice
The repertoire of anti-NP antibodies elicited in CD19TG mice was
examined by generating hybridomas from splenocytes of individual
CD19TG mice immunized with NP.sub.18 -CGG. The hybridomas were
labeled TG2, TG3, TG7, or TG18 depending on the day of splenocyte
isolation post-immunization (Table 3). Splenocytes from mice
boosted with NP.sub.18 -CGG on day 15 were used to generate TG18
hybridomas. The TG2, TG3, TG7 and TG18 fusions generated 164, 36,
210, and 275 hybridomas total with 3, 1, 31, and 10 monoclonal
hybridoma lines isolated that secreted antibodies reactive with
NP-BSA but not BSA in ELISA assays. Surprisingly, none of these
hybridomas secreted .lambda.1-bearing antibodies (Table 3).
From the TG2 and TG3 fusions, three of the four hybridomas (75%)
secreted .mu., .kappa. antibody products, while one (1) produced a
.gamma.2a, .kappa. antibody (Table 3). The relative
affinities/avidities of anti-NP antibodies were determined by
calculating the ratio of NP.sub.5 -binding antibody concentrations
to NP.sub.25 -binding antibody concentrations as previously
described (Herzenberg et al., 1980). The affinity threshold for
IgG1 antibody binding to each NP-BSA conjugate was determined using
monoclonal antibodies with known affinities for NP. The
H33L.gamma.1 antibody (IgG1, .lambda.1; K.sub.a =2.0.times.10.sup.7
M.sup.-1) bound equally well to both NP.sub.5 -BSA and NP.sub.25
-BSA conjugates (binding ratio .about.1.0), whereas the
B1-8.gamma.1 monoclonal antibody (IgG1, .lambda.1) with a
Ka=10.sup.6 M.sup.-1 exhibited 5-fold lower relative binding to
NP.sub.5 -BSA than to NP.sub.25 -BSA (ratio .about.0.2). The
H50G.gamma.1 monoclonal antibody (IgG1, .lambda.1) with a
Ka=1.2.times.10.sup.5 M.sup.-1 did not bind NP.sub.5 -BSA at
detectable levels, but bound NP.sub.25 -BSA (ratio <0.01). Based
on this analysis, these anti-NP antibodies generated from CD19TG
mice had relatively low affinity/avidity values for NP.
From the TG7 fusion, 22 of the 28 hybridomas studied (79%) secreted
.mu. antibodies while the rest secreted .lambda.1 antibodies (Table
3). None of the antibodies bore .lambda.1 light chains, while 68%
bore .lambda.2, 21% bore .lambda.3, and 11% bore .kappa. light
chains. The relative affinities/avidities of the TG7 antibodies
were quite heterogeneous, although the IgG1 antibodies were
generally of lower affinity (Table 3). By contrast, all 10
hybridomas from the TG18 fusion secreted G1, .kappa. antibodies
with affinities/avidities equal to that of the
H33L.gamma.1/.lambda.1 control antibody (Table 3).
Sequence Analysis of Anti-NP Antibodies from CD19TG Mice During
Primary Responses
The heavy chain genes of the TG2, TG3 and TG7 hybridomas were
sequenced by PCR amplification of cDNA made from hybridoma RNA.
Eight of 31 antibodies (26%) were encoded by VDJ rearrangements
containing V.sub.H gene segments common in the anti-NP B cells of
C57BL/6 mice, V186.2, V23, and C1H4 (Table 3). Three of these
antibodies, TG7-14, -17, and -99, may have arisen from a common
progenitor since each had identical VDJ sequences. These were the
only antibodies to display canonical VDJ sequences for anti-NP
antibodies with the preferred YYGS motif in CDR3 (Table 3). Two of
the three V23 containing antibodies, TG7-26, and -170, also shared
identical VDJ sequences. The V.sub.H regions used by all eight
hybridomas were free of somatic mutations. Thus, CD19TG mice are
capable of generating antibody heavy chains typical of those
obtained in Igh.sup.b mice to NP, although these represented a
minority of the antibodies and none of these heavy chains paired
with .lambda.1.
The remaining 74% of primary-response antibodies used V.sub.H genes
not normally found in NP-specific B cells from C57BL/6 mice (Table
3). Sixteen of these antibodies used V.sub.H segments encoded by
known members of the J558 family; 86.22 (1 hybridoma), G4D11 (1
hybridoma), V130 (5 hybridomas, 2 were related), 671.5 (8 related
hybridomas), and C1A4 (1 hybridoma). Remarkably, the majority of
V.sub.H regions did not contain somatic mutations. One anti-NP
antibody, TG7-83, used a previously unidentified V.sub.H segment
similar to the 5D3 gene (Kaartinen et al., 1988) of the J558 family
although six nucleotide differences at the 5' end were homologous
with the 186.2 V.sub.H gene sequence. This V.sub.H sequence is
similar to that of the dC5 antibody from a C57BL/6 mouse (GenBank
accession number AF045488) and the germline V.sub.H II gene, H30,
isolated from BALB/c mice (Schiff et al., 1985) and is therefore
likely to represent a heretofore unidentified V.sub.H gene segment
in C57BL/6 mice.
TG7 hybridomas utilized V.sub.H gene segments from the 7183, Q52
and IX gene families (Table 3). The TG7-3 antibody was encoded by a
novel V.sub.H 7183 family member most similar to the V.sub.H 61-1P
gene of BALB/c mice (Chukwuocha et al., 1994). A C57BL/6 V.sub.H
gene that only differs from TG7-3 at four positions was identified,
although these differences are unlikely to represent somatic
diversification since these residues are found in other VH7183
family members of C57BL/6 mice. The TG7-50, -108, and -110
hybridomas generated unrelated antibody products using V.sub.H
segments almost identical to the OX-2 gene segment, a V.sub.H Q52
family member of BALB/c mice (Lawler et al., 1987). The TG7-125
hybridoma utilized a V.sub.H region identical to the BALB/c
germline OX-1 V.sub.H gene, another member of the Q52 family. The
TG7-118 hybridoma utilized a V.sub.H region most homologous with
the VGAM3-8 V.sub.H gene of C57BL/6 mice, an IX gene family member
(Winter et al., 1985). The TG7-188 V.sub.H sequence was also 98%
homologous with V.sub.H regions of two hybridomas, 5G6 and 264,
from C57BL/6 mice (GenBank accession number AF045504, Nottenburg et
al., 1987). The TG7-188 V.sub.H segment may therefore represent a
new member of the IX gene family in C57BL/6 mice. The AGTC changes
at the 5' end may represent somatic diversification since we were
unable to detect similar sequences in the C57BL/6 genome.
Considerable diversity in D.sub.H and J.sub.H use by all of the
hybridomas was apparent (Table 3), but relatively few antibodies
contained the CDR3 YYGS motif typical of anti-NP antibodies in
C57BL/6 mice. All J.sub.H 1 sequences were of the b allotype. Thus,
the antibody response in CD19TG mice was quite diverse by day 7
after primary immunization, although the hybridomas primarily used
germline genes with no, or few, somatic mutations (Table 3).
Sequence Analysis of Anti-NP Antibodies from CD19TG Mice During
Secondary Responses
All ten hybridomas generated from the TG18 fusion produced
antibodies paired with .kappa. light chains (Table 3). One of
these, TG18-43, carried a member of the J558 family, V23, bearing 2
point mutations. The TG18-161 hybridoma V.sub.H gene segment
matched the V23 sequence except for 4 nucleotide differences; 3
were clustered at codons 9, 10 and 11 which were identical to the
V186.2 gene. Thus, the TG18-161 heavy chain gene rearrangement may
be derived from a hybrid of two well characterized V.sub.H gene
segments, V23 and V186.2, or from a previously unknown J558 family
member. Consistent with the second possibility is the full identity
of the TG18-161 V.sub.H gene with that present in the 70.1.4
hybridoma derived from a C57BL/6 mouse (GenBank accession number
AF006576). Of the remaining eight TG18 hybridomas, six were
clonally related (Table 3) with V.sub.H gene segments homologous
(96%) to the germline 22.1 V.sub.H gene of the J606 V.sub.H family
in BALB/c mice (Brodeur and Riblet, 1984; Hartman and Rudikoff,
1984). The two remaining hybridomas, TG18-5 and TG18-259, shared
identical VDJ sequences with V.sub.H segments encoded by OX2-like
genes of the Q52 family. These hybridomas utilized a V.sub.H gene
segment that differed from those utilized by the unrelated TG7-50,
-108 and -110 hybridomas at three positions that are potential
sites of hypermutation. D.sub.H and J.sub.H gene utilization was
also diverse among the TG18 hybridoma set (Table 3), although none
of the antibodies encoded the YYGS motif. Thus, the repertoire of
the anti-NP antibody response of CD19TG mice substantially diverges
from the response of wildtype Igh.sup.b mice with only 20% (2/10)
of the TG18 antibodies encoded by members of the J558 V.sub.H gene
family and none carried the .lambda.1 light chain.
.lambda. Light Chain Utilization by Anti-NP Antibodies
In contrast with the expanded V.sub.H repertoire used to generate
anti-NP antibodies in CD19TG mice, there was a striking deficiency
in .lambda.1 utilization and the .lambda. light chain repertoire
was remarkably compressed (Table 3). .lambda.2 and .lambda.3
diversification did not occur since only two different .lambda.
light chains were used by sixteen .lambda.-producing hybridomas.
There was no evidence of junctional diversity in any of the light
chains and only one mutation was found in all of the .lambda.3
light chains. Although heavy chain gene mutations are commonly
greater than ten-fold more frequent than .lambda. mutations during
anti-NP responses (Cumano and Rajewsky, 1986; Ford et al., 1994),
the lack of diversity and mutations in this large panel of
antibodies was unexpected.
V.sub.H Utilization by CD19KO Mice
The repertoire of anti-NP antibodies elicited in CD19KO mice was
also examined by generating hybridomas from splenocytes of
individual mice. CD19KO mice were immunized with NP.sub.18 -CGG on
day 0, boosted on day 7, with hybridomas generated on day 10. The
KO10 fusions generated 615 hybridomas total. Only six clonal
hybridomas were isolated (<1%) that secreted .mu., .kappa.
antibodies with low affinities/avidities for NP.sub.25 -BSA, but
not BSA, in ELISA screens (Table 3). Four antibodies were encoded
by non-canonical, germline V.sub.H genes of the J558 family (Table
3). Of interest, the KO10-613 antibody was encoded by a newly
described member of the J558 family, L350-7 (Kasturi et al., 1994).
Two identical antibodies were encoded by a new member of the IX
V.sub.H gene family. Thus, there was little affinity maturation or
selection for canonical sequences in CD19-deficient mice.
Affinity Analysis of Anti-NP Antibodies
Antibodies representing most NP-specific hybridomas from primary
and secondary responses were purified and used for NP affinity
determinations by fluorescence quenching (Azuma et al., 1987; Eisen
and McGuigan 1971; Jones et al., 1986). This assay measures
antibody binding to NP-caproate, a monovalent derivative of the
immunizing hapten. Under the conditions utilized the assay was
sensitive to 7.0.times.10.sup.3 M.sup.-1, below which NP-specific
binding was not detected. Of eight TG7 antibodies, the K.sub.a s
ranged between a low of 7.2.times.10.sup.3 M.sup.-1 for TG7-125 and
a high of 1.6.times.10.sup.5 M.sup.-1 for TG7-180, with an average
affinity of 7.2.times.10.sup.4 M.sup.-1 (FIG. 4). The KO10-613
antibody had a Ka of 1.3.times.10.sup.4 M.sup.-1. By contrast, all
three TG18 antibodies had relatively high affinities, 1.9 to
2.9.times.10.sup.6 M.sup.-1.
The K.sub.a s of the NP-specific hybridomas antibodies were also
compared with K.sub.a s of antibodies generated by transfectomas
producing canonical anti-NP antibodies and representative
antibodies utilized by B cells isolated from NP-specific foci or
germinal centers according to art-recognized techniques. On
average, the NP-specific antibodies generated in CD19TG mice were
of lower affinity than canonical anti-NP antibodies represented by
the B1-8.gamma.1 control antibody (FIG. 4). However, the TG18
antibodies were uniformly of higher affinity than canonical
antibodies or antibodies isolated from germinal centers. Thus, the
CD19TG mouse generates noncanonical anti-NP antibodies of higher
affinity than canonical antibodies generated in wild-type C57BL/6
mice.
B Cell Apoptosis in CD19TG and CD19KO Mice
Apoptosis regulates the immune response of B lymphocytes and
influences selection for affinity maturation within germinal
centers. Mice that constitutively express Bcl-x.sub.L in B cells
exhibit expanded use of non-canonical anti-NP antigen receptors
following immunization with NP-CGG. To assess whether decreases in
apoptosis caused the dramatic shift in repertoire utilization
observed in mice expressing differing CD19 levels, B cell apoptosis
was assessed in situ and in vitro by two methods, TUNEL analysis
and by determining the frequency of hypodiploid B cells labeled
with propidium iodide. Immunohistological analysis of spleen tissue
sections from CD19TG and CD19KO mice revealed that the overall
frequency of apoptotic cells within follicles of each mouse strain
were not obviously different from wildtype C57BL/6 littermates. The
frequency of apoptotic cells in CD19KO mice was increased above
C57BL/6 controls, but the majority of apoptotic cells were within
the T cell zones of the knockout animals. When B cells were
purified from these mice and assessed for the frequency of
hypodiploid B220.sup.+ B cells labeled with propidium iodide,
0.19.+-.0.03% apoptotic B cells were observed in CD19TG B cells,
0.27% in CD19KO B cells, compared with 0.20.+-.0.03% apoptotic B
cells from wildtype littermates. Culturing the B cells overnight
(16 h) or for 2 days in the presence of varying concentrations of
anti-IgM antibodies did not reveal dramatic differences in the
frequency of apoptotic cells in CD19TG mice and controls. By
contrast, the frequency of apoptotic cells was significantly
reduced in CD19KO B cells. Therefore, the repertoire expansion
observed in CD19TG mice does not appear to result from a
significantly reduced rate of B cell apoptosis.
Reactivity of Anti-NP Antibodies with Autoantigens
Since tolerance to self antigens may influence the diversity of the
B cell repertoire and CD19TG mice have defects in peripheral
tolerance, the potential for non-canonical anti-NP antibodies to
react with self antigens was assessed. Of interest was that 27 of
the 47 antibodies analyzed in this study had Arg residues located
within their CDR3 regions compared with 12 of 45 NP-specific
antibodies generated in C57BL/6 mice that were chosen randomly from
the GenBank database. The facts that CD19TG mice produce anti-DNA
autoantibodies and that anti-DNA autoantibodies commonly contain
Arg residues within their CDR3 regions (Krishnan et al., 1995;
Shlomchik et al., 1990) prompted an assessment of whether the
anti-NP antibodies produced by CD19TG mice also reacted with ssDNA.
The TG7-83 IgG1 antibody reacted strongly with ssDNA, at a level
similar to that of serum from autoimmune MRL.sup.lpr/lpr mice (FIG.
5A). In fact, the relative binding of the TG7-83 antibody for ssDNA
was comparable with the binding of two well-characterized,
isotype-matched anti-ssDNA autoantibodies (Krishnan et al., 1995;
Tillman et al., 1992) over a range of antibody concentrations (FIG.
5B). Two additional antibodies, TG2-354 and TG2-417, also bound
ssDNA at levels significantly higher than non-specific control
antibodies. The KO10-613, TG3-471, TG7-75, and TG7-68 antibodies
also reacted with self protein antigens. This finding indicates
that alterations in tolerance regulation in CD19TG mice in part
account for the expanded NP-specific antibody repertoire of CD19TG
mice, in accordance with the methods of the present invention.
Materials and Methods
Mice
The generation of CD19-deficient mice (CD19KO) and human CD19
(hCD19) transgenic mice (CD19TG, h19-1 line, C57BL/6) has been
described in the art (Engel et al., 1995; Zhou et al., 1994). B
lymphocytes of the h19-1 line of CD19TG mice express 3-fold higher
levels of total cell surface CD19 (Sato et al., 1996; Sato et al.,
1997) and have 9-14 copies of the hCD19 transgene integrated into a
single (or closely linked) genomic site(s) on chromosome 7. The
h19-1 mice used in this study were backcrossed with C57BL/6 mice
(Jackson laboratory, Bar Harbor, Me.) for 8 to 10 generations
without a diminution of hCD19 expression and all mice expressed
similar levels of cell-surface hCD19. CD19KO mice were backcrossed
with C57BL/6 mice for 8 to 10 generations. Flow cytometric analysis
demonstrated that B lymphocytes from all mice expressed the
IgM.sup.b but not IgM.sup.a allotype, and the mice only produce
antibodies of the b allotype. All mice were 2-3 months of age at
the time of use and were housed under identical conditions in a
specific pathogen free barrier-facility. All studies and procedures
were approved by the Animal Care and Use Committee of Duke
University.
Antigens and Immunizations
Succinic anhydride esters of (4-hydroxy-3-nitrophenyl)acetyl (NP;
Genosys Biotechnologies, The Woodlands, Tex.) were reacted with CGG
(Sigma Chemical Co., St. Louis, Mo.) or bovine serum albumin (BSA,
Sigma Chemical Co.) as described (Jacob et al., 1991). The coupling
ratio of each hapten/protein conjugate was determined
spectrophotometrically. Eight-week old mice were immunized with a
single intraperitoneal injection of 50 pg NP.sub.18 -CGG conjugate
precipitated in alum (Jacob et al., 1991).
Quantification of Serum Anti-NP Antibody Levels
Serum IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, .kappa. light chain, and
.lambda.1 light chain antibodies specific for NP were quantified by
ELISA. Wells of 96-well flat bottom plates (Costar, Cambridge,
Mass.) were coated with either 5 .mu.g/ml NP.sub.5 -BSA or
NP.sub.25 -BSA in 0.1 M borate-buffered saline (pH 8.4) at
4.degree. C. overnight before the wells were blocked with
phosphate-buffered saline (pH 7.4) containing 2% gelatin and 1%
BSA. Serially-diluted mouse sera were then added to each well at
room temperature for 1.5 hours. After washing with Tris-buffered
saline (pH 7.5) containing 0.05% Tween 20 (Sigma Chemical Co.),
alkaline phosphatase (ALP)-labeled goat antibody specific for mouse
IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, or .kappa. light chain
(Southern Biotechnology Associates, Birmingham, Ala.) was added and
incubated at room temperature for 1.5 hours. .lambda.1 light
chain-bearing antibody binding was assessed using biotinylated
Ls136 (anti-.lambda.1) monoclonal antibody (Reth et al., 1978) and
ALP-conjugated streptavidin (Southern Biotechnology Associates).
ALP activity was visualized using p-nitrophenyl phosphate substrate
(Southern Biotechnology Associates) and optical densities were
determined at 405 nm.
The concentrations of IgM, IgG1, .lambda.1, or .kappa. anti-NP
antibodies were estimated by comparisons to standard curves
generated using serially diluted control monoclonal antibodies on
each plate. The standard for IgG1 and .lambda.1 anti-NP antibodies
was H33L.gamma.1, as is known in the art. The standard for IgM was
B1-8, an IgM anti-NP monoclonal antibody (Reth et al., 1978). The
.kappa. antibody standard was TG18-43 (Table 3). The standard for
IgG2a, IgG2b, IgG3, IgA anti-NP antibodies was serially diluted
serum from a C57BL/6 mouse obtained 10 days following immunization
with NP.sub.18 -CGG.
Enzyme-Linked Immunospot Assays
The frequency of NP-specific antibody-forming cells (AFC) from
single-cell splenocyte and bone marrow suspensions were estimated
by enzyme-linked immunospot (ELISpot) assays using NP.sub.5 -BSA
and NP.sub.25 -BSA conjugates as has been described in the art.
Immunofluorescence Staining and Flow Cytometry Analysis
Single cell suspensions of mouse splenocytes were incubated with
anti-FcgRI/RII monoclonal antibody (clone 2.4G2, PharMingen, San
Diego, Calif.) for 10 min on ice to block Fc.gamma. receptor
function. To determine the frequency of GL7.sup.+ cells among
B220.sup.+ B cells, splenocytes were subsequently incubated with
FITC-labeled GL7 monoclonal antibody (PharMingen),
phycoerythrin-conjugated anti-B220 monoclonal antibody (RA3-6B2,
Caltag, South San Francisco, Calif.), and 7-aminoactinomycin D
(Molecular Probes Inc., Eugene, Oreg.) for 30 min on ice before
washing. The cells were subsequently analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, Calif.). The percentage of
GL7.sup.+, B220.sup.+ cells was calculated from live lymphocytes
selected by forward-side scatter patterns and exclusion of
7-aminoactinomycin D. In additional experiments, splenocytes were
incubated with FITC-labeled anti-B220 antibody (RA3-6B2) and
biotinylated Ls136 antibody, washed, and incubated with
phycoerythrin-conjugated streptavidin. After washing, the frequency
of .lambda.1.sup.+ cells among viable B220.sup.+ lymphocytes was
determined by flow cytometry.
Immunohistochemistry
Six-.mu.m-thick frozen spleen sections were mounted on
poly-L-lysine (Sigma Chemical Co.)-coated slides, fixed, and stored
at -80.degree. C. as described (Jacob et al., 1991). Frozen
sections were stained with horseradish peroxidase (HRP)-conjugated
peanut agglutinin (PNA; ICN Pharmaceuticals, Costa Mesa, Calif.)
and biotinylated Ls136 antibody, followed by
streptavidin-peroxidase (Southern Biotechnology Associates)
treatment as described (Jacob et al., 1991). Additional serial
frozen sections were also stained with HRP-conjugated PNA and
biotinylated GL7 antibody, followed by streptavidin/alkaline
phosphatase, or stained with HRP-conjugated PNA and ALP-labeled
anti-.kappa. light chain antibodies. HRP and ALP activities were
visualized using 3-aminoethyl carbasole (Sigma Chemical Co.) and
naphthol AS-MX phosphate/fast blue BB (Sigma Chemical Co.),
respectively (Jacob et al., 1991). Terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL;
MEBSTAIN Apoptosis kit; Immunotech, Westbrook, Me.) was used to
identify apoptotic cell nuclei (Gavrieli et al., 1992) and the
sections were counterstained with hematoxylin.
Hybridoma Generation
Seven to eight-week-old mice were immunized with single
intraperitoneal injections of 100 .mu.g of NP.sub.18 -CGG conjugate
precipitated in alum on day 0. Two, three, or seven days after
immunization, splenocytes from one or two immunized mice were fused
with nonsecreting P3X63-AgB.653 myeloma cells as described (Kearney
et al., 1979) and subdivided into ten 96 well tissue culture
plates. One CD19TG mouse was boosted on day 15 with the same
antigen and its splenocytes were fused with the myeloma cell line
on day 18. Two CD19KO mice were boosted on day 7 with 100 .mu.g of
NP-CGG conjugate and their splenocytes were fused with myeloma
cells on day 10. The hybridomas generated from these fusions were
named based on the splenocyte source and the fusion day following
immunization: for example, KO10 hybridomas were generated from
splenocytes of CD19KO mice 10 days after the first
immunization.
Monoclonal hybridomas secreting anti-NP antibodies were identified
by ELISA. Culture supernatant fluid from each hybridoma was added
to NP.sub.45 -BSA-coated 96-well flat bottom ELISA plates. After
washing, ALP-labeled goat anti-mouse IgM+IgG+IgA antibodies were
added to each well and ALP activity was visualized using
p-nitrophenyl phosphate substrate. Hybridomas generating
BSA-reactive antibodies were identified by ELISA using BSA-coated
plates and were eliminated. The class and isotypes of NP-reactive
antibodies were determined by ELISA with NP.sub.45 -BSA coated
plates and by using mouse monoclonal antibody isotyping kits
(Amersham Life Sciences, Arlington Heights, Ill.). Hybridomas
secreting .lambda. light chain antibodies were identified by ELISA
using plates coated with goat anti-mouse whole Ig antibodies and
ALP-labeled goat anti-mouse .lambda. light chain antibodies
(Southern Biotechnology Associates) as the developing reagent.
Hybridomas secreting .lambda. light chain antibodies were
identified by immunohistochemical staining using cytospin
preparations of each hybridoma. Hybridomas were centrifuged onto
glass slides, dried for 2 hours, then fixed with acetone at
4.degree. C. for 10 min. The slides were stained with biotinylated
Ls136 antibody, followed by incubation with HRP-conjugated
streptavidin which was visualized as above.
Some NP-specific hybridomas were grown in miniPerm bioreactors
(Heraeus, South Plainfield, N.J.). Their culture supernatant fluid
was concentrated and the antibody product was purified over protein
G-Sepharose (Pierce, Rockford, Ill.) or mannose-binding columns
(Pierce, Rockford, Ill.). Antibody protein concentrations and
purity were determined by light absorption and by antibody
isotype-specific sandwich ELISA.
V.sub.H and Light Chain Gene Utilization
Cytoplasmic RNA was extracted from 0.1-1.times.10.sup.6 hybridoma
cells using the RNeasy Mini Kit (Qiagen Chatsworth, Calif.). First
strand cDNA was synthesized from cytoplasmic RNA using oligo-dT
primers (dT.sub.18) and a Superscript Kit (Gibco BRL, Gaitherburg,
Md.). One .mu.l of cDNA solution was used as template for PCR
amplification of V.sub.H genes. PCR reactions were carried out in a
100-.mu.l volume of a reaction mixture composed of 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 200 .mu.M dNTP (Perkin
Elmer, Foster City, Calif.), 50 pmol of each primer, and 5 U of Taq
DNA polymerase (ISC Bioexpress, Kaysville, Utah). Amplification was
for 30 cycles (94.degree. C. for 1 min, 58.degree. C. for 1 min,
72.degree. C. for 1 min; Thermocycler, Perkin Elmer).
V186.2-related V.sub.H genes were amplified using a sense primer
complementary to the 5' region of the V186.2 gene (primer V186.2;
5' TCTAG AATTC AGGTC CAACT GCAGC AGCC 3'--SEQ ID NO:1) and
antisense primers complementary to the C.mu. coding region (primer
C.mu.-in; 5' GAGGG GGAAG ACATT TGGGA AGGAC TG 3'--SEQ ID NO:2) or
the C.gamma. region (primer C.gamma.1; 5' GAGTT CCAGG TCACT GTCAC
TGGC 3'--SEQ ID NO:3). V.sub.H genes not amplified using the V186.2
primer were amplified using a promiscuous 5' V.sub.H primer
(MsV.sub.H E; 5' GGGAA TTCGA GGTGC AGCTG CAGGA GTCTGG 3'--SEQ ID
NO:4) as previously described (Kantor et al., 1996). A light chain
cDNA was amplified using a V.lambda. primer (5' AACTG CAGGC TGTTG
TGACT CAGGA ATC--SEQ ID NO:5) and a C.lambda. primer (CGGGA TCCGC
TCTTC AGAGG AAGGT GGAAA CA--SEQ ID NO:6). Amplified PCR products
were purified from agarose gels using the QIAquick gel purification
kit (Qiagen) and were sequenced directly in both directions using
an ABI 377 PRISM DNA sequencer after amplification using the Perkin
Elmer Dye Terminator Sequencing system with AmpliTaq DNA polymerase
and the same primers for initial PCR amplification.
Sequences were compared with known V.sub.H sequences using the
BLAST search program provided by the National Center for
Biotechnology Information. V.sub.H genes belonging to the J558
family were analyzed as described (Bothwell et al., 1981; Gu et
al., 1991).
Anti-NP Antibody Affinity Measurements
The K.sub.a s of purified anti-NP antibodies was determined by
fluorescence quenching as described (Azuma et al., 1987; Eisen and
McGuigan, 1971; Jones et al., 1986). Briefly, K.sub.a s for NP and
NIP haptens were measured by fluorescence quenching in a Shimadzu
RF-3501 fluorospectrophotometer (Shimadzu Scientific Instruments,
Columbia, Md.). Excitation and emission wavelengths were 280 and
340 nm, respectively: temperature (25.degree. C.) and pH (7.4) were
held constant. Titration was conducted by adding NP- or
NIP-caproate (Cambridge Research) over a three-log range (10.sup.-8
-10.sup.-5 M) to a known concentration of antibody (33 mM in 2.5 ml
PBS) in quartz cuvettes. Emission signal loss, or quench, versus
antigen concentration was plotted according to the Scatchard
equation to derive association constants for half-maximal
binding.
Anti-ssDNA ELISA
Calf thymus DNA (Sigma Chemical Co.) was purified by repeated
phenol/chloroform extraction followed by ethanol precipitation. The
DNA suspension was boiled for 10 min before immersion in an ice
bath to generate ssDNA. ELISA assays were carried out in 96 well
Immulon II microtiter plates (Dynatech Laboratories, Chantilly,
Va.) that were coated overnight at 4.degree. C. with ssDNA (5 mg/ml
in 0.1 M Na citrate buffer containing 0.15 M NaCl, pH 8.0). Plates
were washed three times with PBS (pH 7.3). Supernatant fluid from
individual hybridomas was diluted (generally 1:2) in PBS containing
1% BSA (Sigma) and 0.05% Tween-20 and added to triplicate wells of
the antigen-coated ELISA plates. Sera from individual MRL
autoimmune mice were used as positive controls which were diluted
(1:100) in PBS containing 1% BSA (Sigma Chemical Co.) and 0.05%
Tween-20. Peroxidase-conjugated goat anti-mouse Ig antibody diluted
in PBS containing 1% BSA and 0.05% Tween-20 was added to the wells
for 1 h before washing three times with PBS. Substrate solution
containing 0.015% 3,3', 5,5'-tetramethylbenzidine (Sigma Chemical
Co.) and 0.01% H.sub.2 O.sub.2 in 0.1 M Na citrate buffer (pH 4.0)
was added at room temperature for 30 min before the OD of the wells
were determined at 380 nm wavelength on a Titertek Plate Reader
(Flow Laboratories, McLean, Va.). OD values within the linear range
of the ELISA were determined using a standard serum obtained from
MRL.sub.lpr/lpr mice with linear regression analysis.
Assessment of Apoptosis
B cells were purified from single cell splenocyte suspensions by
removing T cells with anti-Thy1.2 antibody-coated magnetic beads
(Dynal, Inc., Lake Success, N.Y.). B cell suspensions were analyzed
by flow cytometry following isolation to assess purity. B cell
preparations from CD19KO and C57BL/6 mice were >95% B220.sup.+
while preparations from CD19TG mice were >75% B220.sup.+. B
cells were seeded in 24-well flat bottom plates (Costar) at
1.times.10.sup.6 cells per well with various concentration of
F(ab').sub.2 fragments of goat anti-mouse IgM antibodies (Cappel,
Durham, N.C.) and cultured for 16 hrs or 48 hrs in a CO.sub.2
incubator. Cultured B cells were washed with PBS containing 0.2%
BSA and TUNEL.sup.+ cells were detected by flow cytometry analysis
using the MEBSTAIN Apoptosis kit (Immunotech). The frequency of
apoptotic cells was calculated: % apoptosis=[(% TUNEL.sup.+
cells/(% TUNEL.sup.+ cells+% live cells)].times.100. Cultured cells
were also washed with PBS containing 0.2% BSA followed by PBS
containing 1% glucose and fixed with ice-cold 70% ethanol
overnight. The fixed cells were stained with 0.05 mg/ml propidium
iodide (PI; Sigma chemical Co.) solution containing 100 U/ml RNase
A (Sigma Chemical Co.). Stained cells were analyzed by flow
cytometry and cells with hypodiploid nuclei were considered
apoptotic.
Data Analysis
All data are shown as mean values.+-.SEM unless indicated
otherwise. Analysis of variance (ANOVA) was used to analyze the
data, and the Student's t test was used to compare sample means.
The paired Student's t test was used to compare the means of %
apoptotic cells.
TABLE 3 NP.sub.25 V.sub.H Somatic CDR3 Hybridoma Isotype
ELISA.sup.a NP.sub.5 /NP.sub.25 Family V.sub.H gene.sup.b Mutation
D.sub.H J.sub.H Length TG2-354 M, .kappa. 0.16 <0.01 J558 86.22
0 SP2.3 1 8 TG2-403 M, .kappa. 0.45 <0.01 J558 186.2 0 SP2.2 2 7
TG2-417 M, .kappa. 2.76 <0.01 J558 G4D11 0 FL16.1 2 12 TG3-471
G2a, .kappa. 0.17 <0.01 J558 C1H4 0 SP2.4/6 2 11 TG7-3 M,
.kappa. >3.00 <0.01 7183 (61-1P) .sup. ND.sup.c ND 3 5 TG7-13
M, .lambda.2 2.80 <0.01 J558 V23 0 Q52 2 10 TG7-14 M, .lambda.2
>3.00 0.86 J558 186.2 0 FL16.1 2 10 TG7-17 M, .lambda.2 >3.00
0.29 J558 186.2 0 FL16.1 2 10 TG7-26 G1, .lambda.2 1.39 <0.01
J558 v23 0 Q52 2 10 TG7-32 M, .lambda.2 >3.00 0.53 J558 130 0
FL16.1 2 10 TG7-50 M, .lambda.2 2.42 0.42 Q52 OX2 ND Q52 4 10
TG7-68 G1, .lambda.2 1.40 <0.01 J558 130 5 FL16.1 1 8 TG7-75 G1,
.lambda.2 1.46 <0.01 J558 130 0 FL16.1 1 8 TG7-80 M, .lambda.3
>3.00 1.21 J558 671.5 0 SP2.4/6 2 10 TG7-83 G1, .lambda.2 1.43
0.01 J558 (5D3) ND SP2.6/7 3 5 TG7-88 M, .lambda.2 >3.00 1.31
J558 671.5 0 SP2.4/6 2 10 TG7-93 M, .lambda.3 >3.00 0.98 J558
671.5 0 SP2.4/6 2 10 TG7-99 M, .lambda.2 >3.00 0.92 J558 186.2 0
FL16.1 2 10 TG7-108 M, .lambda.2 >3.00 0.07 Q52 OX2 ND ST4 4 10
TG7-110 M, .lambda.3 >3.00 <0.01 Q52 OX2 ND FL16.1 4 9
TG7-112 M, .lambda.3 >3.00 0.96 J558 671.5 0 SP2.4/6 2 10
TG7-114 M, .lambda.2 >3.00 0.45 J558 130 0 FL16.1 2 10 TG7-118
G1, .kappa. 1.20 0.04 IX (VGAM3-8) ND SP2.5/7 2 8 TG7-125 M,
.lambda.2 >3.00 0.02 Q52 OX-1 ND SP2.9 4 8 TG7-129 M, .lambda.3
>3.00 1.04 J558 671.5 0 SP2.4/6 2 10 TG7-137 G1, .kappa. 0.99
0.04 J558 C1A4 0 FL16.1 4 11 TG7-138 M, .lambda.2 >3.00 0.51
J558 ND ND ND ND ND TG7-159 M, .lambda.2 >3.00 1.06 J558 671.5 0
SP2.4/6 2 10 TG7-167 M, .lambda.3 >3.00 1.38 J558 671.5 0
SP2.4/6 2 10 TG7-170 M, .lambda.2 >3.00 0.05 J558 V23 0 Q52 2 10
TG7-180 M, .lambda.2 >3.00 0.79 J558 130 2 FL16.1 1 12 TG7-186
M, .lambda.2 >3.00 1.01 J558 671.5 0 SP2.4/6 2 10 TG18-5 G1,
.kappa. 1.45 0.97 Q52 OX2 ND SP2 4 7 TG18-41 G1, .kappa. 1.49 1.20
J606 22.1 ND FL16.1 2 10 TG18-43 G1, .kappa. 1.35 0.90 J558 V23 2
FL16.1 2 11 TG18-48 G1, .kappa. 1.58 1.14 J606 22.1 ND FL16.1 2 10
TG18-61 G1, .kappa. 1.64 1.06 J606 22.1 ND FL16.1 2 10 TG18-99 G1,
.kappa. 1.64 0.97 J606 22.1 ND FL16.1 2 10 TG18-161 G1, .kappa.
1.54 1.29 J558 (V23) ND Q52 2 9 TG18-223 G1, .kappa. 1.59 1.14 J606
22.1 ND FL16.1 2 10 TG18-252 G1, .kappa. 1.60 0.82 J606 22.1 ND
FL16.1 2 10 TG18-259 G1, .kappa. 1.52 0.73 Q52 OX2 ND SP2 4 7
KO10-613 M, .kappa. 0.20 <0.01 J558 L350-7 ND SP2.2-5 4 10
KO10-678 M, .kappa. 0.75 0.16 J558 VGAM3.0 0 FL16.1 3 13 KO10-683
M, .kappa. 0.18 <0.01 IX (VGAM3.8) ND FL16.1 3 10 KO10-863 M,
.kappa. 0.55 0.03 J558 vmn2 0 SP2.8 4 12 KO10-1006 M, .kappa. 0.48
<0.01 J558 86.22 1 DST4 3 10 KO10-1121 M, .kappa. 0.15 <0.01
IX (VGAM3.8) ND FL16.1 3 10 .sup.a Values represent mean ELISA OD
results obtained using hybridoma culture supernatant fluid. A
positive control IgM antibody (B1-8, 10 .mu.g/ml) generated mean OD
values of 1.18 while an IgG1 antibody (H33Lyl, 1.0 .mu.g/ml)
generated OD values of 1.76. All OD values were significantly
greater (p < 0.05) than those obtained with control culture
media (IgM ELISA, 0.072 .+-. 0.001; IgG ELISA 0.077 .+-. 0.001) or
supernatant fluid from isotope-matched negative # control
hybridomas. These results are representative of those obtained in
at least three experiments. .sup.b Parenthesis indicate that the
V.sub.H genes used are similar to those cited, but are likely to be
distinct genes. .sup.c ND, not determined because the homologous
gene has not been identified in C57BL/6 mice, the size of the D
region was too small, or there were ambiguities in the sequence of
TG7-138.
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SEQUENCE LISTING (1) GENERAL INFORMATION: (l) APPLICANT: TEDDER,
THOMAS F. (ii) TITLE OF INVENTION: ANTIBODY PRODUCTION METHODS
RELATING TO DISRUPTION OF PERIPHERAL TOLERANCE IN B LYMPHOCYTES
(iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A)
ADDRESSEE: JEFFREY L. WILSON (B) STREET: SUITE 1400, UNIVERSITY
TOWER, 3100 TOWER BOULEVARD (C) CITY: DURHAM (D) STATE: NORTH
CAROLINA (E) COUNTRY: USA (F) ZIP: 27707 (v) COMPUTER READABLE
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COMPUTER: IBM PC/XT/AT compatible (C) OPERATING SYSTEM: Windows 95
(D) SOFTWARE: WORD PERFECT 8 and ASCII (vi) CURRENT APPLICATION
DATA: (A) APPLICATION NUMBER: TO BE ASSIGNED (B) FILING DATE: TO BE
ASSIGNED (C) CLASSIFICATION: TO BE ASSIGNED (vii) PRIOR APPLICATION
DATA: N/A (A) APPLICATION NUMBER: N/A (B) FILING DATE: N/A (viii)
ATTORNEY/AGENT INFORMATION: (A) NAME: JEFFREY L. WILSON (B)
REGISTRATION NUMBER: 36,058 (C) REFERENCE/DOCKET NUMBER: 180/95
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (919) 493-8000
(B) TELEFAX: (919) 419-0383 (2) INFORMATION FOR SEQ ID NO: 1: (l)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 bases (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: 1: tctagaattc aggtccaact gcagcagcc 29 (2)
INFORMATION FOR SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 27 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
gagggggaag acatttggga aggactg 27 (2) INFORMATION FOR SEQ ID NO: 3:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 3: gagttccagg tcactgtcac tggc 24
(2) INFORMATION FOR SEQ ID NO: 4: (l) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 31 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
gggaattcga ggtgcagctg caggagtctg g 31 (2) INFORMATION FOR SEQ ID
NO: 5: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 bases (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 5: aactgcaggc tgttgtgact caggaatc
28 (2) INFORMATION FOR SEQ ID NO: 6: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 bases (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:
6: cgggatccgc tcttcagagg aaggtggaaa ca 32
It will be understood that various details of the invention may be
changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 6 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Mus sp. <400> SEQUENCE: 1
tctagaattc aggtccaact gcagcagcc 29 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Mus sp. <400>
SEQUENCE: 2 gagggggaag acatttggga aggactg 27 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Mus sp. <400>
SEQUENCE: 3 gagttccagg tcactgtcac tggc 24 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 31
<212> TYPE: DNA <213> ORGANISM: Mus sp. <400>
SEQUENCE: 4 gggaattcga ggtgcagctg caggagtctg g 31 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211>
LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Mus sp.
<400> SEQUENCE: 5 aactgcaggc tgttgtgact caggaatc 28
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Mus sp. <400> SEQUENCE: 6 cgggatccgc tcttcagagg aaggtggaaa ca
32
* * * * *